Methods, tip assemblies and kits for introducing material into cells

ABSTRACT

Methods, tip assemblies and kits are provided for introducing material into cells. The tip assemblies include an attachment portion, a channel portion, and a constriction that function to reduce fluid pressure as a fluid passes through the constriction portion from the channel portion, whereby the tip assemblies form pores in the membranes of cells and introduce material into the cells. The material includes for example one selected from the group of: an inorganic compound, a drug, a genetic material, a protein, a carbohydrate, a synthetic polymer, and a pharmaceutical composition.

RELATED APPLICATIONS

The present application is a continuation of U.S. utility applicationSer. No. 16/159,185 filed Oct. 12, 2018, now U.S. Pat. No. 10,364,441,issued Jul. 30, 2019, which is a continuation of U.S. utilityapplication Ser. No. 14/687,020 filed Apr. 15, 2015, now abandoned,which is a continuation of U.S. utility application Ser. No. 13/231,592filed Sep. 13, 2011, now U.S. Pat. No. 9,017,991 issued Apr. 28, 2015which claims the benefit of U.S. provisional application Ser. No.61/438,824 filed Feb. 2, 2011, and international applicationPCT/US2010/027104 filed Mar. 12, 2010 which claims benefit of U.S.provisional application Ser. No. 61/159,856 filed Mar. 13, 2009, each ofwhich hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Methods, tip assemblies and kits are provided for introducing materialinto cells. The material introduced into cells includes an inorganiccompound, a drug, a genetic material, or a pharmaceutical composition.

BACKGROUND

Induced expression of genetic material, for example DNA sequences, inliving cells is the foundation for molecular genetics and molecularbiology. In this process, genetic material encoding for example forgenes, is artificially introduced into the nucleus of a cell, so thatthe cell generates the product of the genes in the form of non-native ormodified proteins, as is achieved by genetic engineering. Inmicrobiology, the method by which one or more particular genes isaltered in a recipient cell is referred to as transformation. Ineukaryotic biology this method is sometimes referred to as transfection,or the introduction of a foreign cloned gene or cDNA into the eukaryoticgenome. Viral or plasmid vectors are the vehicle for the introduction ofDNA sequences.

Many methods have been employed to introduce genetic material into thenucleus or cytoplasm of living cells. These methods have limitations andrequire the conveyance of plasmid cDNA across plasma membranes, whichare the heterogeneous bilayers of lipid molecules found on all cells. Awell-known method to introduce genetic material into the nucleus orcytoplasm of living cells is electroporation, which relies on dielectricbreakdown of the membrane producing gaps of up to 120 nanometers (nm) indiameter (metastable aqueous pores) in the membrane through whichgenetic material enters the cell through electrodiffusion. Anothermethod is the use of transfection reagents including lipid or fat-basedreagents (i.e., lipofectamine) which are essentially detergents thatassociate with DNA, thereby permitting the DNA to pass though the plasmamembrane.

Biolistic transfection is yet another method, and involves a “gene gun”that fires the genetic material coupled to gold nanoparticles at highpressure through the plasma membrane. Mechanical injection is yetanother method that typically uses glass needles that physicallypuncture the plasma membrane to deliver the genetic material usinghydrostatic pressure injection directly into the nucleus.

Efficiencies of these methods have been found to have limitations,particularly due to toxicity, injury, or death to the cells. Thetransfection efficiency of such methods typically is very low orvariable, depending upon the method and the cell type, and is related tothe loss of viability of the treated cells, or the inability of themethod to get the genetic material through the cell membrane. Forexample, transfection efficiency of the gene gun to treat a variety ofdifferent cell lines was observed to be successful with only about 1% to4% of treated cells. Lipofection yields successful recombinants in onlyabout 10% to 20% of treated cells. Polycationic lipid reagents generallydo not exceed 40% efficiency. Most importantly, certain cell types arenot amenable to any method, including certain types of in rune systemcells, human stem cells, muscle cells, nerve cells, and other cell typesthat do not divide and are therefore maintained in culture only asprimary cells.

A more biological approach relies on virus-mediated transfection forintroduction of genetic material into cells. This approach utilizesspecific engineered viral vectors derived from strains such asadenovirus, sindbis virus, retrovirus, baculovirus or lentivirus, etc.to infect the cells. The viral genomes are engineered to carry thegenetic sequences of interest, and are consequently a relativelytime-consuming and labor-intensive method compared to mechanical meansof introducing genetic material. This approach has a significantmethodological limitation in that the time delay or lag to obtainprotein expression depends not only on the efficiency of the cell totranscribe and translate the genetic material, but also on the infectionefficiency of the virus. In addition, use of live virus requires speciallaboratory safety standard conditions, i.e., biohazard level 2.

Infection efficiency depends on many variables, and viral-mediatedengineering is characterized by a lag of at least a day or several daysto observe protein expression in cell populations. Furthermore,infection efficiency is cell-type specific, i.e., particular viruses donot infect certain cells.

There remains a need for a rapid, efficient, and universal method forintroducing material such as genetic material into any type of cell,including cell types that have heretofore been difficult or impossibleto transfect with any commercially available method.

SUMMARY

An aspect of the invention provides a tip assembly for introducing acomposition in a fluid into cells, the assembly including: an attachmentportion open to the atmosphere and proximally fitted to a flow devicethat generates at least one of a positive pressure and a negativepressure for impelling or directing the fluid; a channel portioncontiguous to and distal to the attachment portion and the flow device;a constriction portion contiguous with the channel portion, such that aconstriction portion inner diameter and a constriction portion crosssectional area respectively are smaller than a channel portion innerdiameter and a channel portion cross sectional area respectively, and adistal end of the constriction portion has an opening for ejecting ordrawing the fluid, such that increased fluid velocity and a decreasedpressure in the fluid in the constriction portion compared to velocityand pressure in the channel portion enhances formation of membrane poresin the cells, so that the tip assembly introduces the composition intothe cells through the membrane pores.

An embodiment of the tip assembly is disposable, alternatively, the tipassembly is reusable. In various embodiments, the tip is at least oneselected from: translucent, transparent, modular, washable andsterilizable.

In various embodiments of the tip assembly, at least one of theattachment portion, the channel portion, and the constriction portionincludes a substance selected from the group of: a glass, a metal, aplastic, a polymer, a nano-based composition, a composite materialcomprising at least two different types of substances, and the like. Anano-based composition includes: a nano-metal, a nano-ceramic, anano-polymer, and the like. For example, tip assembly composed of thecomposite material is for example a glass and a polymer or a plastic anda polymer.

In an embodiment of the tip assembly, at least one of the attachmentportion, the channel portion, and the constriction includes a surfacecomponent that prevents cell adherence, for example, a wax or a polymer.

The tip assembly in an embodiment has a distal end of the constrictionthat is non-lacerative, for example the distal end is diamond polished,heat-polished, chemically polished or flame-polished.

In an embodiment of the tip assembly, an inner diameter of theattachment portion of the tip assembly fits the flow devicerespectively, in a male to female arrangement. The tip assembly in anembodiment has an outer diameter of the attachment portion of the tipassembly respectively, that fits the flow device in a male to femalearrangement, respectively. In an embodiment of the tip assembly, theattachment portion is smooth. Alternatively, the attachment portionincludes a ridge, a protrusion or an indent. In various embodiments ofthe tip assembly, the attachment portion connects to a flow device bysliding, twisting, or rotating.

In various embodiments of the up assembly, the attachment portion of thetip assembly that fits the flow device is selected from a pipette, asyringe, a compressor, or a pump. The compressor is a fluid compressoror an air compressor for impelling the fluid. In an embodiment of thetip assembly, and the pump includes a fluid pump or a pressure pump.

In embodiments of the tip assembly the channel portion cross sectionalarea or the constriction portion cross sectional area is bounded by acircle, an ellipse, a rectangle or a square. For example the channelportion is a rectangular channel and the constriction portion is acylinder.

In an embodiment of the tip assembly, a distal end of the constrictionportion has an inner diameter that is less than or substantially equalto an inner diameter of the opening. In various embodiments, the tipassembly includes or encompasses a volume selected from the group ofabout: 2 microliters (μl), 20 μl, 50 μl, 200 μl, 500 μl, 1 milliliter(ml), 5 ml, and about 10 ml. Greater volumes such as 50 ml, 100 ml and500 ml are also envisioned as within the scope of the invention. Invarious embodiments, the channel portion inner diameter is about 1.0millimeter (mm) to about 10.0 mm, and the dimension of the constrictioninner diameter is about 0.05 mm to about 2.0 mm, such that theconstriction inner diameter is smaller than the channel portion innerdiameter.

In an embodiment of the tip assembly, the channel portion curvature andconstriction inner curvature characterize a fluid path for the fluidflowing through the tip assembly which includes a formula. For example,the formula represented in two dimensions isf(x)=p1x ⁷ +p2x ⁶ +p3x ⁵ +p4x ⁴ +p5x ³ +p6x ² +p7x+p8wherein x is a radial distance from a center axis on the fluid path toan inner surface of the tip and coefficients with 95% confidence boundsin parentheses include: p1 is −2.611e⁻¹⁶ (−6.043e⁻¹⁶, 8.206e⁻¹⁷), p2 is3.954^(e−13) (−3.195^(e−13), 1.11^(e−12)), p3 is −1.845^(e−10)(−7.821^(e−10), 4.131^(e−10)), p4 is 1.662^(e−08) (−2.394^(e−07),2.726^(e−07)), p5 is 7.537^(e−06) (−5.186^(e−05), 6.694^(e−05)), p6 is−0.002137 (−0.009375, 0.005101), p7 is −0.003185 (−0.4114, 0.4051), andp8=268.6 (261, 276.3). Alternatively, the formula represented in twodimensions isf(x)=p1x ⁸ +p2x ⁷ +p3x ⁶ +p4x ⁵ +p5x ⁴ +p6x ³ +p7x ² +p8x+p9such that coefficients with 95% confidence bounds in parenthesesinclude; p1 is 2.285 (1.388, 3.182), p2 is 3.465 (1.782, 5.149), p3 is−15.68 (−20.04, −11.32); p4 is −20.38 (−27.24, −13.52); p5 is 44.27(36.5, 52.04); p6 is 53.96 (45.69, 62.23); p7 is −58.72 (−64.02,−53.42), p8 is −123.5 (−126.4, −120.6), and p9 is 186.5 (185.6, 187,4).The formula is various embodiments includes a formula in two dimensionsthat is substantially similar to formulaf(x)=p1x⁷+p2x⁶+p3x⁵⁻⁵+p4x⁴+p5x³+p6x²+p7x+p8 and formulaf(x)=p1x⁸+p2x⁷+p3x⁶+p4x⁵+p5x⁴+p6x³+p7x²+p8x+p9. For example the formulagraphed on two dimensions lies between the two formulae above andresults in the tip assembly functionally introducing the composition ina fluid into a cell the same as the formulae.

In an embodiment, the tip assembly curvature has a shoulder extendinglaterally from an outward surface of the attachment portion, for examplethe shoulder functions in manually removing the tip assembly from theflow device, or a lower ejector section of the flow device removes thetip assembly from the flow device programmably or manually. For examplethe shoulder includes a geometry selected from the group including: acylinder, a cone, a rectangular, a regular polygon and a square.

The channel portion of the tip assembly has a length that is a functionof the volume of fluid, or is a function of a peak velocity of fluidnecessary for forming the pores in the cells. The channel portiongenerally has a marking or a plurality of markings for visuallyidentifying or measuring the volume in the tip assembly.

An aspect of the invention provides a method for introducing acomposition in a fluid into cells, the method involving: contacting thecells in a reservoir with a fluid including the composition; inserting atip assembly into the reservoir, the tip assembly including: anattachment portion open to the atmosphere and proximally fitted to aflow device that generates at least one of a positive pressure and anegative pressure for directing the fluid; a channel portion contiguousto and distal to the attachment portion and the flow device; aconstriction portion contiguous with the channel portion, such that aconstriction portion inner diameter and a constriction portion crosssectional area are smaller than a channel portion inner diameter and achannel portion cross sectional area, a distal end of the constrictionportion having an opening for ejecting or drawing the fluid, such thatincreased fluid velocity and a decreased pressure in the fluid in theconstriction portion compared to velocity and pressure in the channelportion enhances formation of membrane pores in the cells; and,passaging a mixture of the cells, the fluid and the composition at leastonce through the tip assembly using a flow device that generates atleast one of a positive pressure and a negative pressure, so thatpassaging the mixture forms the membrane pores in the cells andintroduces the composition into the cells.

The cells passaged in the tip assembly are prokaryotic cells or areeukaryotic cells.

In various embodiments, the composition is at least one selected fromthe group of: an inorganic compound, a drug, a genetic material, aprotein, a Carbohydrate, a synthetic polymer, and a pharmaceuticalcomposition. For example the pharmaceutical composition is at least oneagent selected from: an anti-tumor, an antiviral, an antibacterial, ananti-mycobacterial, an anti-fungal, an anti-proliferative and ananti-apoptotic. For example, the composition is a vector carrying anucleic acid that encodes a protein or peptide having an amino acidsequence.

In various embodiments, the method further includes after passaging,observing localization of the composition to at least one subcellularcompartment or cellular structure of a cell selected from: a nucleus, amitochondrion, a Golgi body, a chloroplast, a chromoplast, an axon, acytoplasmic membrane, a nuclear membrane, an endosome, a vesicle, avacuole and a cytoplasm. For example the method in various embodimentsfurther includes after passaging, observing localization of thecomposition to the nucleus of the cells, or observing localization ofthe composition to cytoplasm of the cells.

In an embodiment of the method, observing the localization furtherincludes visualizing the composition with a detectable marker, forexample the detectable marker is selected from the group consisting of:detectable, fluorescent, colorimetric, enzymatic, radioactive, and thelike. For example, the detectable marker is a green fluorescent proteinor a cyanine 3 fluorescent dye.

In various embodiments of the method, observing the localization furtherincludes quantifying directly the product of the composition thatentered the cell from the group consisting of: mRNA, siRNA, shRNA,microRNA, DNA, RNA, and protein.

In an embodiment of the method, passaging the mixture further includesredirecting the mixture at least once to the reservoir. In an embodimentof the method, passaging the mixture further involves dispensing themixture into a receptacle, or into one or more of a plurality ofreceptacles. In an embodiment of the method, passaging the mixturefurther involves dispensing the mixture into a constricted channelcontinuous with the constriction at the distal end of the tip.

In an embodiment of the method, the cells that are a population are aplurality of cells. The cells in general are living cells, and themethod further involves measuring cell viability and observing that thecell viability is not substantially reduced. In various embodiments ofthe method the cell viability is at least about: 1%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of control cells not socontacted and/or passaged through the tip assembly. For example cellviability is determined by at least one method such as contacting thecells with propidium iodide, observing cell morphology using amicroscope, and measuring cell attachment by resistance or impedance bymeasuring real-time cell electronic sensing (RT-CES) in a multi-cellculture dish or E-plate.

In an embodiment of the invention, passaging the mixture in the tipassembly reduces the pressure at the constriction portion at least about0.05%, 1%, 5%, 10%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, or 95% in contrast to the mixture passaged through the channelportion only.

In various embodiments of the method, prior to contacting, the methodfurther includes preparing the fluid. For example, the fluid includes aCa⁺² concentration less than about 500 nanomolar (nM), about 200nanomolar (nM), about 150 nM, about 100 nM, about 75 nM, or less thanabout 50 nM. In various embodiments of the methods, the fluid includes aMg⁺² concentration of at least about 0.5 millimolar (mM), at least about1 mM, at least about 2 mM, at least about 5 mM or at least about 10 mM.In various embodiments of the method, the fluid includes a magnesiumconcentration of less than about 200 nanomolar (nM), about 150 nM, about100 nM, or about 75 nM.

An embodiment of the method further includes after passaging,centrifuging the mixture to obtain a cell pellet and a supernatant. Forexample, the method further includes removing the supernatant, addingcell culture medium to the reservoir, re-suspending the cell pellet inthe medium and culturing the cells.

An embodiment of the method uses cells that are living postmitoticcells. In various embodiments of the method, the cells include at leastone cell type selected from the group consisting of: epithelial cells,hematopoietic cells, stem cells, spleen cells, kidney cells, pancreascells, liver cells, neuron cells, glial cells, smooth or striated musclecells, sperm cells, heart cells, lung cells, ocular cells, bone marrowcells, fetal cord blood cells, progenitor cells, tumor cells, peripheralblood mononuclear cells, leukocyte cells, and lymphocyte cells. In anembodiment of the method, the cells include physiologically inactivecells, for example the physiologically inactive cells are selected fromthe group of: inhibited, UV-inactivated, enucleated, anucleate, andheat-killed. In an embodiment of the method, the cells includenon-reproducing cells or synthetic cells having an artificial membrane.

The method in various embodiments involves the composition that is agenetic material including a DNA or an RNA. In various embodiments ofthe method, the RNA is at least one selected from the group of: mRNA,tRNA, rRNA, siRNA, RNAi, miRNA, and dsRNA or a portion thereof. In anembodiment of the method, the DNA includes cDNA. In an embodiment, themethod further includes observing transfection or transformation of thecells.

The method in various embodiments further includes applying to themixture at least one selected from the group of: an electric field, alight comprising at least one wavelength, and a sound pulse. Forexample, the electric field, the light, or the sound pulse is used tofurther stabilize the cells in the mixture, activate the composition, orto further enhance the size or duration of the membrane pores in thecells for introducing the composition into the cells.

In various embodiments of the method, passaging is performed over aperiod of time, for example at least about 1 second, about 2 seconds,about 4 seconds, about 6 seconds, about 8 seconds, about 10 seconds,about 30 seconds, about 1 minute, about 3 minutes, about 5 minutes,about 10 minutes, about 15 minutes, about 20 minutes, or about 30minutes. For example, the passaging is performed continuously over aperiod of time. Alternatively, the passaging is performed intermittentlysuch that the period of time includes a plurality of passaging and alsoa plurality interruptions or pauses. For example, the period ofpassaging and/or the period of pauses is milliseconds. In variousembodiments, the plurality of passaging and the plurality of pauses isabout 2, about 5, about 10, about 15, about 20, about 25, about 30,about 40, about 50, about 75, and about 100 seconds.

An aspect of the invention provides a kit for introducing a compositionin a fluid into cells the kit having a tip assembly for passaging amixture of the cells and the fluid including: an attachment portion opento the atmosphere and proximally fitted to a flow device that generatesat least one of a positive pressure and a negative pressure forimpelling the fluid; a channel portion contiguous to and distal to theattachment portion and the flow device; a constriction portioncontiguous with the channel portion, such that a constriction portioninner diameter and a constriction portion cross sectional area aresmaller than a channel portion inner diameter and a channel portioncross sectional area, such that a distal end of the constriction portioncomprises an opening for ejecting or drawing the fluid, such thatincreased fluid velocity and a decreased pressure in the fluid in theconstriction portion compared to velocity and pressure in the channelportion enhances formation of membrane pores in the cells, whereby thetip assembly introduces the composition into the cells through themembrane pores; and a container.

In an embodiment, the kit further includes a reservoir or a receptacle.In an embodiment, the kit further includes instructions for use, forexample, at least one instruction including: contacting the cells in areservoir with the fluid including the composition; inserting the tipassembly into the reservoir; contacting the attachment portion of thetip assembly to a flow device that generates at least one of a positivepressure and a negative pressure; and passaging a mixture of the fluidand the composition at least once through the tip assembly using theflow device, whereby passaging the mixture forms the membrane pores inthe cells and introduces the composition into the cells. In anembodiment of the kit, the instructions include methods for obtaining ordirecting fluid into the reservoir or the receptacle.

The kit in various embodiments further includes one or more of: atransfection agent, a buffer, or a medium for at least one cell, cellline, or cell strain. In an embodiment, the kit further includes a flowdevice that applies at least one of a positive pressure and a negativepressure for directing or impelling the fluid.

An aspect of the invention provides a system for introducing compositionin a fluid into cells, the system having a flow device that generates atleast one of a positive pressure and a negative pressure for impellingthe fluid; and, a tip assembly for passaging a mixture of the cells andthe fluid including: an attachment portion that connects to the flowdevice, a channel portion contiguous to and distal to the attachmentportion and the flow device, a constriction portion contiguous with thechannel portion, such that a constriction portion inner diameter and aconstriction portion cross sectional area are smaller than a channelportion inner diameter and a channel portion cross sectional area, suchthat a distal end of the constriction portion includes an opening forejecting or drawing the fluid, such that increased fluid velocity and adecreased pressure in the fluid in the constriction portion compared tovelocity and pressure in the channel portion enhances formation ofmembrane pores in the cells, whereby the system introduces thecomposition into the cells through the membrane pores.

The system in an embodiment further includes a receptacle adjacent tothe opening for receiving or retaining the fluid passaged through thetip assembly. In an embodiment, the system further includes a pluralityof receptacle (e.g., tubes, vials, flasks, beakers, and plates) adjacentto the opening for receiving or retaining the fluid passaged. The systemin an embodiment further includes an optical device for detecting thecells and/or the composition, for example a flow cytometer, amicroscope, a mass spectrometer, a UV detector, a spectrophotometer, ora cell counter.

In various embodiments of the system, the tip assembly includes at leastone selected from the group consisting of: a glass, a metal, a plastic,a polymer, a nano-based composition, a composite material that includesat least two different types of substances; and the like.

In an embodiment of the system, the distal end of the constrictionportion is polished and smooth, for example the distal end is heatpolished.

The flow device in various embodiments of the system includes at leastone selected from the group of: a syringe, a plunger, a bulb, adiaphragm, and a compressor.

In an embodiment of the system, the flow device is controlled oroperated manually. In various embodiments of the system, control andoperation of the flow device is at least one selected from: automated,electromechanical, and programmable. For example the system furtherincludes a computer or user interface that controls and operates thesystem. For example the computer is connected to the computer or userinterface using wireless components or using, a connector such as anelectric cord or USB connector.

In various embodiments of the system, control and operation of the flowdevice includes the control of at least one selected from the group of:flow velocity; flow acceleration; mass flow rate; initial velocity ramp;starting velocity; maximum velocity; starting position; inflow velocityramp, outflow velocity ramp, inflow velocity, outflow velocity, cutoffvelocity; period of time the fluid is under pressure generated by theflow device; temperature, and period of time the fluid is held in thechannel portion, constriction portion, or both.

In various embodiments of the system, the flow device generates a flowvelocity along the length of the tip assembly, the flow velocityselected from about: about 0.1 centimeter per second to about 1 cm/s,about 1 cm/s to about 5 cm/s, about 5 cm/s to about 15 cm/s, about 15cm/s to about 20 cm/s, and about 20 cm/s to about 40 cm/s.

In various embodiments of the system, the flow device generates a massflow rate selected from a group consisting of about: 0.01 milliliter perminute (ml/min), 1 ml/min. 10 ml/min, 25 ml/min, 50 ml/min, 100 ml/min,and 150 ml/min.

In various embodiments of the system, the flow device generates a fluidacceleration in the tip assembly of about 0.6 microliters per second persecond (μl/s/s), about 1 μl/s/s, bout 6 μl/s/s, about 10 μl/s/s, about12 μl/s/s, about 24 μl/s/s, about 36 μl/s/s, about 48 μl/s/s, and about60 μl/s/s.

The tip assembly in all embodiment of the system further includes atleast one valve. In various embodiments, the valve is situated orlocated proximal to or in the channel portion, between the channelportion and the constriction portion, in the constriction portion,distal or adjacent to the constriction portion, or adjacent to theopening. For example, the valve is used to introduce at least one of thefluid, the composition or the cells. In an embodiment of the system, theat least one valve includes a plurality of valves, for examplepositioned adjacent to the opening or proximal to the constrictionportion.

The system in various embodiments is a closed system with the openingbeing connected to the valve for example using a conduit, thus forre-circulating the mixture or the fluid from the opening to the channelportion, or for directing the mixture or the fluid from the channelportion to a receptacle.

The system in an embodiment further includes a power source, for examplea battery, power cell, or solar panel.

In an embodiment, the system further includes a connector forinteracting with at least one of: a user interface, a computer, ahand-held device, a transmitter, and a display. For example, thetransmitter connects to a server which then connects to a computer.

The system in an embodiment includes a conduit for connecting the flowdevice to the tip assembly. For example the connector includes tubingfor example plastic or polymer tubing. In an embodiment of the system,the tubing includes at least one filter or membrane for the cells.

In an embodiment the system is located in a housing. For example thehousing is located primarily on a bench or table, or alternatively ismobile. In an embodiment, the housing is located on or within ahigh-throughput sampling or testing device.

The system in an embodiment includes a sensor for sensing the flowdevice or the fluid. In various embodiments of the system, the sensordetects at least one selected from the group of: pressure, time,temperature, flow device signal, flow device current, and flow devicelocation. In embodiments of the system, the sensor communicates with theflow device or a microprocessor.

In an embodiment of the system, the flow device directs iterations ofpassaging the fluid and the cells through the tip assembly. In variousembodiments of the system, the cells include or are at least one celltype selected from the group consisting of: epithelial cells,hematopoietic cells, stem cells, spleen cells, kidney cells, pancreascells, liver cells, heart cells, lung cells, ocular cells, sperm cells,smooth or striated muscle cells, glial cells, neuronal cells, bonemarrow cells, fetal cord blood cells, premitotic cells, progenitorcells, peripheral blood mononuclear cells, leukocyte cells, andlymphocyte cells.

In various embodiments of the system, the composition is at least oneselected from the group of: an inorganic compound, a drug, a geneticmaterial, a protein, a carbohydrate, a synthetic polymer, and apharmaceutical composition. For example, the genetic material includesDNA or RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing and graph showing an exemplary shape of the interiorsurface of a tip assembly for introducing a material such as acomposition in a fluid into a cell.

FIG. 1 panel A is a drawing of a three-dimensional representation of aninner surface of an exemplary tip assembly having a top or proximalopening which is located proximal to source of the material, fluid, andpressure, and a bottom or distal opening which is distal to the sourceof material, fluid, and pressure. The drawing shows that the tipassembly has a channel portion of a greater inner diameter andcross-section area than a distally located constriction portion. Changesin the radial dimension are not shown to scale relative to theproximal-distal dimension to emphasize the geometry of the distal end ofthe tip assembly. The scale bar is 200 micrometers (microns; μm).

FIG. 1 panel B is a curve showing the relationship between the length ofthe tip along the fluid path from the proximal to the distal opening anda radial distance from a center axis on the fluid path to an innersurface of the tip assembly, fitted using the eighth degree polynomialfunction characterizing the curvature of the tip assembly shown in FIG.1 panel A. The radial distance of the inner surface of the tip assemblyfrom a center axis on the fluid path (microns) is shown on the ordinateand the length of the tip along the fluid path (microns) is shown on theabscissa. The fluid path along the inner surface of the tip from theproximal (top) opening channel portion and constriction to the distal(bottom) opening is shown from left to right.

FIG. 1 panel C is a photograph of an exemplary tip of a tip assembly forintroducing material in a fluid in a cell, with a smaller tip diameter(175-200 μm), and a constriction portion of length 250 μm proximal tothe distal opening of the tip assembly. The tip assembly was made fromcustom fabricated glass tube having an ID of 0.88 mm, OD of 1.23 mm andwall thickness of 0.14 mm.

FIG. 1 panel D is a drawing of a three-dimensional representation of anexemplary tip as shown in FIG. 1 panel C. The drawing shows that the tipopening is narrower than the opening of the tip shown in FIG. 1 panel A.The drawing also shows a constriction potion, 250 μm long proximal tothe distal end of the tip assembly. Scale bar is 500 μm.

FIG. 2 is a drawing and graph showing an exemplary shape of a tipassembly for introducing material in a fluid into a cell.

FIG. 2 panel A is a drawing of a three-dimensional representation of theinner surface an exemplary tip assembly having a top or proximal openingwhich is located proximal to source of the material, fluid, andpressure, and a bottom or distal opening which is distal to the sourceof material, fluid, and pressure. The drawing shows that the tipassembly has a channel portion of a greater inner diameter andcross-section area than a distally located constriction portion. Changesin the radial dimension are not shown to scale relative to theproximal-distal dimension to emphasize the geometry of the distal end ofthe tip assembly. The scale bar is 200 micrometers μm.

FIG. 2 panel B is a curve showing the relationship between the length ofthe tip along the fluid path from the proximal to the distal opening andthe radial distance from a center axis on the fluid path to the innersurface of the tip, fitted using the eighth degree polynomial functioncharacterizing the curvature of the tip assembly shown in FIG. 2 panelA. The radial distance of the inner surface of the tip assembly from acenter axis on the fluid path (μm) is shown on the ordinate and thelength of the tip along the fluid path (μm) is shown on the abscissa.The fluid path along the inner surface of the tip from the proximal(top) opening channel portion and constriction to the distal (bottom)opening is shown from left to right.

FIG. 3 is a set of photographs showing cells photographed underdifferent detection conditions. The photograph on the left is aphotograph of a brightfield microscope field of view showing acollection of ciliary ganglion neurons carrying a gene for greenfluorescent protein (GFP) introduced by the methods and devices herein,photographed at seven hours after culturing. The photograph on the rightis a photograph of an epifluorescence microscope field of view of thesame ciliary ganglion neurons as in FIG. 3 left, photographed at thesame time.

FIG. 4 panel A is a set of photographs each showing a nonneuronalciliary ganglion cell that is the recipient of a gene encodinggreen-fluorescent protein (GFP), introduced by the methods and devicesherein. The photograph on the left is a brightfield microscope field ofview, and the photograph on the right is an epifluorescence microscopefield of view showing GFP fluorescence as light areas in the cell.

FIG. 4 panel B is a set of photographs each showing a ciliary ganglionneuronal cell that is the recipient of genes encoding GFP, introduced bythe methods and devices herein. The photograph on the top is abrightfield microscope field of view, and the photograph on the bottomis a fluorescence microscope field of view. These data show that GFPexpression is maximal at 48 hours after receiving the GFP gene. Further,GFP expression is abundant in compartments in the cytoplasm and absentfrom the nucleus.

FIG. 5 panel A is a set of photographs each showing a ciliary ganglionneuron that is the recipient of a gene encoding GFP-myosin Va fusionprotein, introduced by the methods and devices herein, photographed attime point eight hours after having the genetic material introduced intothe cell. The photograph on the left is a brightfield microscope fieldof view, and the photograph on the right is an epifluorescence,microscope field of view, each taken at the same point in time. Thephotographs of the cells treated with GFP-myosin Va, gene by methodsdescribed herein show a specific distribution in the neuronal processestypical of native myosin protein.

FIG. 5 panel B is a set of photographs each showing the same ciliaryganglion neuron from the same culture as shown in FIG. 3 panel A,photographed at 28 hours after the genetic material was introduced intothe cell. The photograph on the left is a brightfield microscope fieldof view, and the photograph on the right is an epifluorescencemicroscope field of view, each taken at the same point in time. Thesephotographs show localization of myosin Va to each of the cell body andthe neuronal processes, particularly the branch points (b) and the tipsof the growing axonal processes (indicated in the immunofluorescentphotographs by asterisks).

FIG. 6 is a set of photomicrographs and bar graphs showing effects entryof genetic material by methods described herein observed on fluorescenceabundance of the tips of neuronal projections (growth cones), andfilopodial length, respectively. Cells were transfected with a each of aconstruct carrying a gene encoding enhanced GFP-myosin Va (myosin-VaGFP), an enhanced GFP-myosin Va that includes only the tail region(myosin-Va tail GFP) and therefore does not bind the actin cytoskeleton,or a control GFP construct (GFP). Cells were transfected and probed forfluorescence after 15 hours in cell culture. Each pair of images of asingle cell in FIG. 6 panel A includes a brightfield (Nomarski ordifferential interference contrast microscopy, DIC) micrograph on theleft and a matched fluorescence image of the same cell on the right,apart from images c and d which are additional examples of growth coneswith abundant enhanced GFP-myosin Va.

FIG. 6 panel A is a set of fluorescence image photomicrographs takenwith a laser scanning confocal microscope (LSCM) of neuronal growthcones transfected with constructs carrying a gene (myosin-Va GFP,myosin-Va tail GFP and GFP control). Each of a construct encoding anenhanced GFP-fusion protein encoding the full-length (FL) neuronalisoform of chicken myosin-Va heavy chain, and a construct encoding atruncated form consisting of the full tail (FT) region that includes theentire IQ motif, were transfected into neurons using a method describedherein. Growth cones of cells transfected with the construct carryingthe gene encoding myosin-Va GFP showed increased abundance of enhancedGFP myosin-Va in the central and peripheral growth cone regions andalone neuronal projections compared to cells transfected with theconstruct carrying the gene encoding myosin-Va mutant tail region or acontrol GFP construct control. These data demonstrate successfultransfection of neuronal cells with the two different myosin Vaprotein-encoding constructs, and also a role of myosin-Va in filopodiallength extension. Scale bar is 10 μm.

FIG. 6 panel B is a set of brightfield DIC micrographs of individualgrowth cones transfected with a construct carrying a gene encodingmyosin-Va GFP, a construct carrying a gene encoding myosin-Va tail GFP,or a construct carrying a gene encoding the GFP control. Growth conestransfected with the gene encoding myosin-Va GFP were observed todisplay significantly longer finger-like membrane projections(filopodia) than growth cones transfected with the control gene encodingGFP. Growth cones tranfected with the construct carrying the geneencoding the mutant tail region of myosin-Va showed significantlyshorter filopodia compared to growth cones transfected with the geneencoding GFP or the gene encoding myosin-Va GFP. Data from thesephotomicrographs demonstrate that successful transfection was observed,and that transfection of the different myosin Va protein variantsresulted in specific and different effects in growth of nerve cellprojections at the growing tips. Scale bar is 10 μm.

FIG. 6 panel C is a set of bar graphs showing filopodial length ofgrowth cones observed at each of eight hours (left) and 15 hours (right)after transfection and plating. Enhanced GFP-myosin-Va overexpressionwas observed to result in significantly increased average filopodiallength at eight hours and 15 hours post-transfection (p<0.01, Student'st-test).

FIG. 7 is a set of pairs of photographs each pair showing untreatedsperm cells (Untreated) on the left, and the same sperm cells that weretransformed by methods and systems herein with a fluid containing aplasmid encoding a variant of green fluorescent protein, the variantentitled pCherry (Shaner N C et al., 2004, Nat Biotechnol22(12):1567-1572), on the right (pCherry) and untreated controls on theleft. Dark field photographs on the left (panels a, c, and e) are takenwith fluorescence microscopy, and on the right (b, d, and f) Nomarskioptics (differential interference contrast microscopy). Panels (a) and(b) show a low power field of view of a group of treated (pCherry) spermcells. Panels (c) to (f) show high power fields of different views ofsperm cells with prominent pCherry fluorescence. Fluorescence intensityin untreated control cells was attributed to background autofluorescence(fluorescence native to the cell). Panels (a) and (b) show a low powerfield of view with many sperm cells. Bright fluorescence points aredebris from other cell types. Panels (c) and (d) show a higher powerfield of view illustrating the background level of nonspecificfluorescence in untreated sperm cells from the same field as in (a) and(b). In the right panel of images (pCherry transfected) sperm cellsusing methods and devices herein show expression of pCherry protein asdemonstrated by significant fluorescence throughout the cells especiallyin the sperm head and midpiece, with a lesser amount observed in theprinciple piece, or sperm tail.

FIG. 8 is a set of photomicrographs showing primary human umbilical veinendothelial cells (HUVEC) 24 hours after a tip assembly was used totransfect cells with a plasmid encoding a fusion protein of enhancedgreen fluorescent protein (EGFP) and human porcine endogenous retrovirusreceptor (HuPAR2). HuPAR2 protein localizes specifically in subcellularmembranous compartments surrounding a cell nucleus. The cells wereplated on a glass-bottom 35 millimeter Petri dish for 15 minutes forattachment to the dish, and were cultured in growth medium at 37° C.

FIG. 8 panel A shows HUVEC cells transfected using a tip assembly withvectors carrying genes encoding a fusion protein of enhanced greenfluorescent protein (EGFP) and HuPAR2. The left photograph shows thecell visualized by differential interference contrast (DIC); the middlephotograph shows the same cell visualized by a fluorescence microscope;and the right photograph shows an overlay of the DIC photograph and thefluorescence microscope photograph. Data show that the tip assemblyeffectively transfected the cell as determined by intense stainingobserved in perinuclear subcellular compartments indicating presence ofthe EGFP-HuPAR2 fusion protein in the cell. Scale bar is 10 μm.

FIG. 8 panel B is a photomicrograph of several HUVEC cells transfectedusing a tip assembly with vectors carrying genes encoding the fusionprotein of EGFP and HuPAR2. The cells were visualized by DIC. Thephotomicrograph shows significant perinuclear staining of GFP-HuPAR2protein in HUVEC cells. The DIC data for these plurality of HUVEC cellstransfected with genes that encode a fusion protein of EGFP and HuPAR2are substantially similar to DIC data in the photograph of the singletransfected HUVEC cell (FIG. 8 panel A, left photograph).

FIG. 9 is a drawing of a three-dimensional representation showing anexemplary shape of a tip assembly having a body including a proximalopening 900 attachment shoulders 901, an attachment portion 902, achannel portion 903, a constriction portion 904, and a distal opening905. The fluid path direction is from the proximal opening 900 to thedistal opening 905 or alternatively from the distal opening 905 to theproximal opening 900. The channel portion 903 was designed to have agreater inner diameter and cross-section area than the constrictionportion 904. The scale bar is 200 μm.

FIG. 10 is a set of photographs of an exemplary transfection system.

FIG. 10 panel A is a photograph of a computer programmable syringe pump101, 102 connected to an external computer by means of a USB converter103 and an RS232 interface 104 configured in the inner side of the metallid 105 of a jump box 106. The RS232 interface 104 to USB converter 103connection is linked by a cable to an external computer for controlusing the data analysis software MATLAB (Mathworks, Natick, Mass.).

FIG. 10 panel B is an enlarged view of the programmable syringe 101 andthe pump mechanism 102 positioned on the jump box 106 right. Theexemplary syringe 101 is borosilicate glass, has a UHMWPE (ultra-highmolecular weight polyethylene) seal 107 which lubricates and is solventresistant, and has a plunger 108. A length of TYGON® tubing 109connected at one end to the syringe is routed through the metal lid 105of the jump box 106 to a connector, and to tubing that attaches directlyto the proximal end of a soda lime glass capillary tube.

FIG. 10 panel C shows a power supply 110 located inside the jump box 106to the lower left.

FIG. 10 panel D is a photograph of the jump box 106 inside a tissueculture flow hood for sterile conditions. The metal lid 105 is in theclosed position, and the outer lid 111 is in an open position. TheTYGON® tube 109 for connecting to a tip extends from the metal lid 105,to the edge 112 of the metal lid 105.

FIG. 11 is a schematic drawing illustrating exemplary movement of thesyringe plunger 108 in fluid cycling with eight cycles. The returnstroke operates according to the same parameters as the executionstroke, i.e. outflow motion has the same characteristics as inflowmotion. A cycle consists of an execution stroke followed by a returnstroke. The position of the plunger location along the length of syringeat a time point during a cycle is represented on the ordinate, and timeis represented on the abscissa. The plunger initially moves fromposition 0 to position 500, the load position, pauses for 200milliseconds (ms), and moves downward to position 400, which reduces thefluid volume to less than an initial fill volume, leaving a cyclingvolume in the tip. After a 100 ms pause the plunger ramps from position400 to 0. Each ramp is shown have a starting velocity, a final velocityand an acceleration from the starting to the final velocity.

FIG. 12 is a set of confocal fluorescence microscope, images of primaryHUVECs (human umbilical vein endothelial cells) transfected with aplasmid using the methods and apparatus herein, encoding a fusionprotein EGFP-CDC42 (enhanced green fluorescent protein-cell divisioncontrol protein 42). Scale bars are 20 μm.

FIG. 12 panel A shows EGFP-CDC42 staining observed 24 hours aftertransfection using laser scanning confocal microscopy. The staining wasobserved in the cytoplasm at the periphery compared to little or nostaining in the nucleus of the cell.

FIG. 12 panel B shows EGFP-CDC42 expression as a result of transfectionin a pair of daughter cells that had recently divided. Expression wasobserved to be concentrated in a region between daughter cells.

FIG. 12 panel C shows a larger magnification of HUVEC cells transfectedwith EGFP-CDC42 at standard PMT (photomultiplier tube) sensitivity.Fluorescence was observed localized to large granules.

FIG. 12 panel D shows HUVEC cells transfected with EGFP-CDC42 in whichthe image was scanned at a higher than standard PMT sensitivity.EGFP-CDC42 expression was observed in subcellular granules smaller insize than granules observed in panel C.

FIG. 13 is a set of confocal fluorescence microscope images and a bargraph showing data obtained from primary HUVEC cells transfected usingthe methods and the devices herein with a 7 kb plasmid encoding anEGFP-actin fusion protein.

FIG. 13 panel A shows a cluster of primary HUVEC cells transfected withEGFP-actin. Transfection and expression of EGFP was observed localizedto actin filaments of the cell cytoskeleton.

FIG. 13 panel B is an image of a primary HUVEC cells transfected withEGFP-actin showing expression of EGFP in the actin filaments of thecell.

FIG. 13 panel C is a bar graph showing EGFP-actin mRNA copy number inHUVEC cells transfected with the methods and devices herein or with airAMAXA Nucleofactor™ (Lonza Cologne GmbH, Cologne, Germany)electroporation unit. TC indicates transfection with the plasmid and CTindicates a control procedure absent the EGFP-actin plasmid. AMAXAindicates data obtained with the AMAXA Nucleofactor™ electroporationunit.

FIG. 14 is a line graph showing quantification of EGFP fluorescence ofJurkat cells transfected with different amounts (2 μg, 5 μg or 10 μg per50 μl fluid volume) of a plasmid encoding EGFP. Cells treated with 5.0μg of plasmid showed greater EGFP fluorescence than the backgroundfluorescence observed in control cells.

FIG. 15 is a set of confocal microscope fluorescence images of primarymouse brain astrocytes transfected using methods and device herein withEMT plasmid or Cy3-labeled miRNA, Scale bar is 10 μm.

FIG. 15 panel A is a confocal fluorescence microscope image of a fewprimary mouse brain astrocytes transfected with EGFP plasmid. The cellwith a round shape has fluorescence intensity near background level.

FIG. 15 panel B is a confocal fluorescence microscope image of a clusterof primary mouse brain astrocytes transfected with EGFP plasmid showingcells with varying levels of fluorescence intensities.

FIG. 15 panel C is a confocal fluorescence microscope image of a groupof primary mouse brain astrocytes transfected with EGFP plasmid showinga cell with high fluorescence intensity surrounded by cells with lowerlevels of fluorescence intensities.

FIG. 15 panel D is an overlay of a confocal fluorescence microscopeimage of a primary mouse brain astrocyte transfected with EGFP plasmidon a brightfield image of the same cell, showing morphological featuresof the cell.

FIG. 15 panel E is a confocal fluorescence microscope image of twoprimary mouse brain astrocytes transfected with Cy3-labeled miRNAshowing internalized miRNA, which is localized mostly in the cytoplasmas tiny round spots.

FIG. 15 panel F is a confocal fluorescence microscope image of a groupof mouse brain astrocytes transfected with Cy3-labeled miRNA showingvarying levels of internalization of miRNA in different cells. TheCy3-labeled miRNA is excluded from the nucleus.

DETAILED DESCRIPTION OF EMBODIMENTS

The invention herein provides methods, devices and kits for introducingmaterial into cells, for example for introducing a composition such as agenetic material into living cells or non-viable cells. The methods,devices and kits herein utilize high fluid velocities, and the examplesherein show that cell survival during the loading process is enhanced,resulting in reliable and highly efficient expression of recombinantproteins in cultured transfected cells.

Cells are surrounded by a plasma membrane comprising a bilayer of lipidsand fat molecules. The integrity of the plasma membrane is affected bythe electrostatic forces among the individual lipid molecules, and byinteractions between the membrane and the underlying cellularcytoskeleton. Individual lipid molecules diffuse through the plane ofinner leaflets and outer leaflets of the bilayer. The behavior of theselipid molecules within the bilayer is described using the fluid mosaicmodel of the plasma membrane that states that the lipid bilayer ischarged and polar to both repel water and prevent the passage of waterinto and out of the cell. The bilayer serves as a barrier also forbiological molecules including nucleic acids, proteins, and fats.

Without being limited by any particular theory or mechanism of action,the methods herein using fluid dynamic technology, in combination withlow Ca⁺² conditions, passaging cells at such high velocity that plasmaand possibly also nuclear membranes are stretched momentarily, therebyproducing transient holes in the membranes that permit entry of plasmidor linear DNA in a molecular shape that is long and relatively thin. Asthe diameter of a single DNA helix is 2 nanometer (nm), and as circularDNA plasmid is subject to coiling and supercoiling, the plasmid acquiresa structural configuration capable of entering a cell through resultingopenings in the cell membrane produced by the methods as describedherein. Electroporation results in openings as great as 120 nm indiameter, in certain examples herein it is envisioned that the methodsherein result in openings in the membrane similar to this size, forexample about 60 nm to about 100 nm, about 100 nm to about 120 nm, orabout 120 to about 140 nm.

The term “cells” includes living cells, non-metabolizing, resting orinhibited cells, and non-reproducing cells. For methods herein, cellsare suspended in a fluid or medium in a container, and a pressuredifferential is applied across the plasma membrane. The pressuredifferential is caused inter alia by different pressures exerted by theionic constituents of the medium, the cytoplasm, and pressure exerted onthe medium. Compressibility of the medium is a factor that affects thepressure differential. Cells are conveniently placed in various types ofmedia, generally in aqueous media which includes biological andnaturally occurring fluids such as blood or tissue. Water is relativelyincompressible, and compressibility is not related to observed pressuredifferences imposed on the cell in aqueous media. Water temperature ifconstant does not affect the fluid density, which also remains constant.This pressure of an incompressible fluid is termed static pressure.

Molecules in a fluid are in constant state of motion and exert pressureon the walls of a container, which is referred to as total pressure. Ifset into directed or ordered motion, a dynamic pressure is producedwhich is an additional type of pressure associated with momentum ofmoving molecules. Therefore motion of molecules contributes to staticpressure, i.e., dynamic pressure. The addition of dynamic pressure tothe static pressure results in a total pressure in a flowing system.Thus, a cell in a fluid is subject to a number of dynamic forces andpressures.

Additional fluid dynamics factors are considered in the circumstances offluid passing in a conduit or tube into a narrowed or constrictedportion. The change in pressure accompanying a change in fluid velocitythrough a tube with a changing cross section area is described by theBernoulli equation which states that if a fluid flowed through areduction in cross-sectional area of a tube an accompanying reduction inpressure and an increase in fluid velocity would be observed. TheBernoulli equation statesv ²/2+gz+p/r=constantin which v is the fluid flow speed at a point in the streamline, g isthe acceleration due to gravity, z is elevation to a point above areference plane with a z direction opposite to the direction ofgravitational acceleration, p is the pressure, and r is the fluiddensity. Bernoulli's principle elucidates a reduction in tube diameterresulting in a decrease in fluid pressure, a process known as theVenturi effect.

The Venturi effect occurs as a result of satisfaction of the law ofconservation of energy, from a fluid passing through a constrictionincreasing the fluid velocity in the constriction to conserve mass. Asthe velocity of the fluid increases so does the kinetic energy.Therefore a decrease in fluid pressure occurs to counteract the increasein kinetic energy.

Cells moving in a fluid-filled channel or passageway undergo an increasein speed in a constriction of the channel, in passing through a narrowerdiameter exit aperture. These cells in suspension experience a suddenchange from high pressure to low pressure. Low pressure results intemporary stretching of the cells, creating temporary holes in cellmembranes, permitting entry of material present in the fluid or solutionsurrounding the cells, including without limitation genetic materialscDNA, siRNA, miRNA. Low Ca⁺² concentration in the fluid or solutionpromotes prolongation of time required for cell membrane sealing,further promoting entry of material down a pressure and concentrationgradient through the holes in the membrane and into the cell interior.The Bernoulli principle is a mechanism by which fluid pressurefacilitates entry of large molecules such as DNA, or other molecules inthe fluid such as smaller molecules including a drug into the cells.

Examples herein utilize a tip assembly that includes a channel portionand a constriction to create pores or holes in a cellular membrane. Anexemplary three dimensional representation of the tip assembly shape isshown in FIG. 1 panel A. Without being limited by any particular theoryor mechanism of action, it is envisioned that after passage of cellsthrough the narrowing or constriction as shown for example in FIG. 1there is a distal opening with curved edges for two-way passaging, inone embodiment an enlargement into a more distal continuation of thechannel into a section, having the same, larger or smaller diameter. Theinside curvature of the shape shown in FIG. 1 panel A is represented intwo dimensions as a cross section (FIG. 1 panel B). The cross sectionsof the tip assembly are parallel to the fluid flow. An exemplarycurvature of the fluid flow and tip assembly shape in one embodimentcharacterized in two dimensions by the following equation:f(x)=p1x ⁸ +p2x ⁷ +p3x ⁶ +p4x ⁵ +p5x ⁴ +p6x ³ +p7x ² +p8x+p9where x is a radial distance from a center axis on the fluid path to aninner surface of the tip, and the coefficients (with 95% confidencebounds in parentheses) are: p1 is 2.285 (1.388, 3.182); p2 is 3.465(1.782, 5.149); p3 is −15.68 (−20.04, −11.32); p4 is −20.38 (−27.24,−13.52); p5 is 44.27 (36.5, 52.04); p6 is 53.96 (45.69, 62.23); p7 is−58.72 (−64.02, −53.42), p8 is −123.5 (−126.4, −120.6); and p9 is 186.5(185.6, 187.4). Parameters applied to obtain goodness of fits were: sumof squares due to error (SSE) is 66.5, r-square (R²) is 0.9997, adjustedR² is 0.9997, and root mean squared error (RMSE) 1.341.

The skilled artisan alters the degree of change in curvature in the tipassembly to change the rate at which the pressure changes during fluidflow. For example, the various channel portions are constructed havingvarious lengths depending on a necessary volume of fluid, fluidpressure, or fluid velocity for introducing material into cells. Forexample, a 2 centimeter (cm) channel portion having a 1 millimeter (mm)inner diameter at the proximal end is constructed to sufficientlyaccelerate 50 μl of fluid through the tip assembly. Thus, curvature ismade steeper or shallower than the embodiment of curvature shown in FIG.1 panels A and B. A shallower curvature is shown in three dimensions inFIG. 2 panel A. The cross-sectional shape of the inside curvature of thefluid path in two dimensions is shown in FIG. 2 panel B. This twodimensional curve is fitted using the eighth degree polynomial equationdenoted and which is shown below:f(x)=p1x ⁷ +p2x ⁶ +p3x ⁵ +p4x ⁴ +p5x ³ +p6x ² +p7x+p8where x is a radial distance from a center axis on the fluid path to aninner surface of the tip, and the coefficients (with 95% confidencebounds in parentheses) are: p1 is −2.611e⁻¹⁶ (−6.043e⁻¹⁶, 8.206e⁻¹⁷); p2is 3.954^(e−13) (−3.195^(e−13), 1.11^(e−12)); p3 is −1.845^(e−10)(−7.821^(e−10), 4.131^(e−10)); p4 is 1.662^(e−08)(−2.394^(e−07),2.726^(e−07)); p5 is 7.537^(e−06)(−5.186^(e−05), 6.694^(e−05)); p6 is−0.002137 (−0.009375, 0.005101); p7 is −0.003185 (−0.4114, 0.4051); andp8=268.6 (261, 276,3). Parameters used to obtain goodness of fit were:SSE is 31.09, R² is 0.9998, adjusted R² is 0.9997, and RMSE is 1.189.

Without being limited by any particular theory or mechanism of action,changes in curvature in the tip assembly are customized for thecharacteristics of the fluid used (e.g., viscosity, temperature, andmolecular components) and also on the type of cell utilized as largercells may required larger constriction sizes. Blood cells for exampleare only about 5 μm to about 10 μm in diameter. In contrast, Xenopuslaevis oocytes are 1000 μm or 1 mm in diameter. Cells having a largersurface area to volume ratio (i.e., smaller cells) generally would beused in methods herein with smaller diameter constrictions and greaterpressure reductions. For example a suitable pressure reduction in thetip assemblies herein includes at least about 0.1%, about 0.5%, 1%, 5%.10%, 15%, 20%, 25%, 30%. 35%,40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,85%, 90%, or about 95%. The pressure drop resulting from the reductionin diameter of the path leading to the distal opening is dependent onvolumetric flow rate and the change in diameter. For example, given agradual reduction in diameter from 1 mm to 0.25 mm, with a flow rate of5 ml/min of water, the pressure drop would be 0.28 millibar (mbar); witha flow rate of 10 ml/min, the pressure drop would be 5.55 mbar; with aflow rate of 10 ml/min, the pressure drop would be 5.55 mbar; with aflow rate of 50 ml/min, the pressure drop would be 27.9 mbar; with aflow rate of 100 ml/min, the pressure drop would be 55.6 mbar; and witha flow rate of 150 ml/min, the pressure drop would be 2643.4 mbar. Thetip assemblies herein are used for any cell type including smaller orlarger cells including animal cells, plant cells, and prokaryotic cells.

The extent of reduction in diameter of a fluid path determines thedecrease in fluid pressure. The manner and degree to which the fluidpressure decreases affects the rate and amount of pore formation in thecellular membrane for a cell moving in the fluid path. FIG. 1 panel Aand FIG. 2 panel A show an exemplary fluid path and channel shape thatnarrows and accordingly decreases the fluid pressure and enhances poreformation in cells in the fluid path. The channel located proximal tothe constriction has a uniform non-varying shape that leads to thedistal narrowing or constriction. The tip assemblies envisioned hereinhave fluid paths or curvatures that are exemplified by the formulasabove.

The tip assemblies are manufactured in various embodiments of any of avariety of suitable materials and suitable aperture sizes. The tip inrelated embodiments is manufactured of a polymer such as a waterrepellent material or coated with an agent to prevent cell adhesion tothe tip assembly or droplet formation that prevents accurate dispensingof the fluid.

Methods for constructing tips include blow molding, vacuum forming, andthermoforming, are described in Smith, U.S. Pat. Nos. 7318,911;6,482,362; and 6,117,394 issued Jan. 15, 2008, Nov. 19, 2002, and Sep.12, 2000; Pelletier et al., U.S. Pat. No. 7,794,664 issued Sep. 14,2010; Taggart et al., U.S. Pat. No. 6,596,240 issued Jul. 22, 2003; andTezuka et al., U.S. Pat. No. 5,136,468 issued Aug. 9, 1994. Borosilicateglass materials and methods are shown for example in Marques, U.S. Pat.No. 7,341,966 issued Mar. 11, 2008 and Watzke et al,, U.S. Pat. No.5,736,476 issued Apr. 7, 1998.

The methods and tip assemblies are suitable for incorporation intoautomated robotic systems, for reliable introduction of DNA into cellsin a rapid and high-throughput manner. For example, the tip assembliesherein are used to transfect and to transduce cells in an automatedmanner to reduce contamination of cells. The tip assemblies herein areenvisioned in various embodiments as attaching to manual pipettes and toautomated pipettes and dispensing systems such as single-tip pipettesystems and multi-tip pipette systems respectively.

Without further limiting the invention, it is here envisioned that oneor more alternative mechanisms may operate to produce the observedtransfection effects. Unlike most existing approaches that rely uponshock, pH, high energy or ultrasonics to affect the energetic of poretransport, these other hypothesized mechanisms rely on cell deformationper se rather than deformation arising from the cell flowing through, anarrowing channel.

Repeated exposure of a cell in fluid suspension to distortion in a waythat the shape of the cell alternates between spherical and oblate maycause rearrangement of the cortical cytoskeleton underlying the plasmamembrane, affecting mobility of lipids or lipid rafts, which caninfluence membrane pore formation. The fluidity of the membrane may beaffected by changes in the underlying cytoskeleton (in a manner similarto that noted in red blood cells by Sventina et, al., 2004,Bioelectrochemistry, 62: 107-113; and gross cellular deformation (suchas flattening of the cell) may contribute to changes in fluidity. Apressure change that might expose the cells to repeatedcompression/expansion independent of fluid flow (ie: a staticsuspension) may potentially also lead to pore formation, since poreformation itself will be dependent upon membrane fluidity. Suchrepetition at a rate substantially lower than existing sonic orultrasonic frequencies, may also be visualized as resulting in aperistalsis of the cell or low-frequency pulsing that may affect thebarrier energy that prevails at a pore under static cellular conditions.To optimize this effect for particular cells and transfection agents,the extent of such effects may be tested, quantified or otherwiseestablished using a different test instrument to introduce a suddenpressure change or sequence of changes within a static fluid column.This flow-free mechanism would be relatively free of potentiallydestructive shear conditions which have been known to damage cells inprior art transfection protocols.

The cultured neurons transfected by methods and devices shown inexamples herein include postmitotic cells that have reached a finaldevelopmental stage and do not further divide. Neurons are fragile, andprior art manipulation techniques, such as electroporation, have beenfound to damage the cells such that regeneration capabilities forphysiological processes and viability in culture are negativelyimpacted.

Primary cultures of certain neurons such as ciliary ganglion neurons anddorsal root ganglion neurons, have typically been used at a stage thatis optimal for experimental manipulation, from one hour to twelve hoursafter culturing. Beyond twelve hours after culturing, the neuronalprocesses become so intertwined and overgrown that even in low densitycultures the ability to monitor neuronal regeneration and outgrowthbecomes increasingly limited with time. Thus, a method to rapidlyintroduce and express genetic material in cells such as neurons remainsa long-felt need. It is envisioned that cell-based experimental systemsthat require rapid expression of genes will benefit from the methods andtip assemblies described herein. Furthermore, high-throughputtransfection required in such processes as protein production can beimproved using the rapid and efficient nature of this method andapparatus. Another application of this method is found in cell-basedgene therapy for clinical use, for example, gene silencing using siRNA.

The methods herein for introducing a composition such as a geneticmaterial into cells provide significant improvements over the prior artbecause the method is rapid, reliable, economical (with no need forexpensive reagents); recipient cells are characterized by very highviability and expression efficiency. Using the prior art method ofelectroporation, the highest efficacy obtained is only about 40% ofrecipient cells which express the nucleic acid. For adenovirus infectionexpression the highest efficacy obtained is about 30% to 85% and isgenerally lower. Gene, guns typically achieve between 1% and 5%efficiency.

In contrast, the methods herein resulted in visible protein expressionin neurons (postmitotitic cells) within three hours post-transfection.In addition, the present method achieved consistently high transfectionefficiencies of 80% to about 100% within 24 hours. Furthermore, themethods herein advantageously do not require viruses, for example,recombinant adenoviruses, which require biosafety level 2 due toinfectiousness. The present method is performed without use of toxicchemicals, complex procedures, or viruses. Cells that are dissociatedfrom tissues or from the surface of a culture plate are suitablematerials for use in the methods herein. Therefore methods herein areapplicable to cells of a tissue obtained by dissociation, and to cellssuspensions e.g., primary cells and cultured cells of a cell line.

The methods herein are applicable also to a tissue in situ, and to amonolayer of cells in culture. The methods herein bypass the relativelylengthy periods of time (several hours or days) required for chemical orviral vectors to express genes for functional analysis. Certain methodssuch as cationic reagents, the gene gun, and electroporation are limitedby the size or length of cDNA that can be used, thereby limiting thetypes of proteins which can be addressed in an experiment or screen.Examples herein include use of a very large plasmid encoding thefull-length protein of myosin Va, which has a molecular weight of about110 kDa. The data in the Examples herein show that constructs ofdifferent sizes, for example DNA encoding proteins that are much largerthan myosin Va, were introduced into cells more effectively andefficiently using the method, tip assembly and kits of the presentapplication than by conventional methods. Introducing the geneticmaterial in Examples herein also includes using a transfection agent ora plurality of transfection agents. For example, the transfection agentis a nanoparticle, a liposome, a viral vector, a bacteriophage, and adetergent. For example, the transfection agent is Lipofectamine.

An exemplary method includes one passage, or a plurality of passages,for example about five to ten passages, that are performed within a oneminute period. For example, a single passage or multiple passages,repeated passages performed at more than one time, are rapidlyperformed. Depending on the cell type, additional embodiments areenvisioned to include passaging five tunes every ten minutes for aspecific period of time, for example, during a time period of about onehour, about two hours, or about five hours.

In general, it was observed that a plurality of passages wasconsistently sufficient to introduce material, e.g., genetic materialand a drug, into a plurality of cells. Cells were maintained in cellculture medium at a suitable temperature, e.g., 37 ° C., after a singletreatment, or between treatments.

The term “introducing” as used herein refers to any of a variety ofmethods for delivering a composition such as a macromolecular or a lowmolecular weight molecule into a cell, either in vitro or in vivo, suchmethods including transformation, transduction, transfection, andinfection. Vectors include plasmid vectors and viral vectors. Viralvectors include retroviral vectors, lentiviral vectors, or other vectorssuch as adenoviral vectors or adeno-associated vectors. Methods forconstructing vectors are shown for example in Ericsson, T. A. et al,2003 PNAS vol. 100 (10): 6759-6764.

The term “passaging”, as used herein means impelling a composition or amixture, for example a solution or a suspension, through at least aportion of a length of the apparatus and tip assembly, i.e., a proximalportion, a middle portion, and a distal portion. In related embodiments,passaging means impelling the fluid entirely through the apparatus,including removing a suspension of cells from a receptacle into the pipeportion and replacing the cells into the same receptacle, ordistributing the cells through the tip assembly portion into a pluralityof receptacles, or into at least one of the plurality of receptacles.

The methods herein were performed on different cell types, some of whichhave previously been categorized as refractory to genetic manipulation.Cell types include for example immortalized cells, actively growingcells, for example, cells in culture, and also postmitotic cells forexample isolated as primary cultures. Examples herein demonstratesuccessful use of the methods with cells that have not in the pastconveniently responded favorably to transfection reagents or toelectroporation, resulting in low viability, or cell-type specificdifferences in transfection efficiency. Further, expression of proteinencoded by the introduced genetic material using the methods herein wasobserved to be significantly more rapid, within three hours of thetreatment, which is at least about a two to three fold faster rate ofexpression of genes compared to the rate observed following introductionof genes by prior art methods.

The myosin-Va constructs described in Examples herein are derived fromthe chicken neuronal isoform of myosin-Va (Espreafico E. M., et al.,1992, J. Cell Biol. 119(6): 1541-1557). A construct pCB6-FL (EGFP-MVawith FL referring to full length) contains the myosin-Va full lengthheavy chain (amino acid residues 1-1830) in a pCB6 plasmid. A truncatedform of MVa consists of the entire last IQ motif (from ARV to SRV, aminoacid residues 880-1830) pEGFP-FT (EGFP-MVaFT, with FT referring to fulltail). These constructs expressed in melanoma cells were shown to haveeffects on melanosome trafficking (da Silva Bizario J. C., et al.,(2002) Cell Motil. Cytoskeleton 51(2): 57-75).

An aspect of the present invention provides a pressurized fluid flowapparatus including: a pipe portion having an inner diameter and anouter diameter and a distal end and a proximal end, such that the distalend is open to the atmosphere, such that the inner diameter of the pipeportion is about 1.0 mm to about 5.0 mm; the apparatus further includingan exit tip assembly adjacent to and contiguous with the distal end ofthe pipe portion, such that the exit tip assembly has an inner diameterand an outer diameter, the inner diameter of the exit tip assembly beingabout 0.1 mm to about 0.5 mm; and a suction device connected to theproximal end of the pipe portion for generating positive pressure andnegative pressure. The exit tip assembly is further optimized inExamples herein.

A related embodiment of the apparatus further includes a receptacleadjacent to the exit tip assembly for receiving and retaining fluidpassaged through the pipe portion and exit tip assembly. An embodimentof the apparatus includes a pipe portion with a diameter that variesalong the length of the pipe portion, for example, from a largerdiameter to a smaller diameter to a larger diameter, the smallerdiameter constituting a constriction of the pipe.

An embodiment of the pipe portion and/or the exit tip assembly includesat least one of from: borosilicate glass, aluminosilicate glass, and thelike. In a related embodiment, the pipe and/or the tip includes flintglass (including lead oxide, titanium dioxide, zirconium dioxide orsimilar metals) and the like. Alternatively, the pipe and/or tipincludes a plastic, such as a polymer.

An embodiment of the apparatus includes the distal end of the exit tipassembly polished by heat treatment to create a non-lacerative surface.For example, heat treatment includes flame-polishing. A non-lacerativesurface of the distal exit opening or hole of the tip assembly minimizesshearing of cells when encountering the edge of the distal exit tipassembly at high velocities, thereby improving efficiency of the processby increasing viability of treated cells.

In general, the suction device generates a flow velocity along thelength of the pipe portion, the velocity selected from at least one of agroup consisting of about: 5 cm/s, 15 cm/s, and 20 cm/s. In anembodiment of the apparatus, the suction device generates a flowvelocity along the length of the pipe portion, the flow velocityselected from at least one of about 5 cm/s to about 10 cm/s, about 10cm/s to about 15 cm/s, about 15 cm/s to about 20 cm/s, and about 20 cm/sto about 40 cm/s.

In general, the suction device generates a mass flow rate at the distalexit of the tip assembly, the flow rate selected from at least one ofabout: 0.03 ml/min, 0.06 ml/min, 0.3 ml/min, 0.5 ml/min, 1.0 ml/min, and2.0 ml/min. In an embodiment of the apparatus, the suction devicegenerates a mass flow rate at the exit tip assembly selected from atleast about: 0.01 ml/min, 0.05 ml/min, 0.1 ml/min, 0.2 ml/min, 0.4ml/min, 0.8 ml/min, 1.2 ml/min, 1.5 ml/min. and about 3 ml/min.

A related embodiment of the apparatus includes the suction device thatgenerates a mass flow rate along the length of the pipe portion selectedfrom at least one of about: 1.0 ml/min, 5 ml/min, 10 ml/min, 50 ml/min,100 ml/min, and 150 ml/min. For example, the suction apparatus generatesa mass flow rate along the pipe portion selected from at least one ofabout 15 ml/min, at least about 25 ml/min, at least about 40 ml/min, atleast about 60 ml/min, at least about 75 ml/min, at least about 90ml/min, or at least about 125 ml/min.

An embodiment of the suction device is manually controlled. The phrase“manually controlled” as used herein means operated directly by theuser, so that at least one of the choices of flow rates and access tocells and receptacles is controlled by the user at the time of use. Inan alternative embodiment, the suction device is automated, i.e., is atleast partially or completely controlled automatically so that the userneed not even be present, for example the device is robotic andoperationally linked to a computer program and computer. A relatedembodiment of the suction device includes at least one device that ismanually controlled, and at least one device that is automated.

An embodiment of the distal exit opening having a tip assembly furtherincludes a constriction in a continuous flow system controlled by atleast one valve, for example, is controlled by two, three, or fourvalves, etc. For example, the at least one valve is a plurality ofvalves positioned laterally proximal to the distal exit hole, withinmillimeters or micrometers proximal to the exit, or all are proximal tothe exit, for example, arrayed circularly around the exit.Alternatively, at least one or more of the plurality of valves is distalto the exit.

An aspect of the present invention provides a method for introducing amaterial such as a protein, a lipid, genetic material, or a drug orother low molecular weight component into a cell, the method including:contacting in a receptacle at least one cell with a compositionincluding an effective amount of the material to obtain a resultingmixture; and, inserting the apparatus of any of the embodiments hereininto the receptacle and passaging the mixture of cells and the materialthrough the apparatus at least once, such that passaging the mixturehaving the cells and the material through the apparatus introduces thematerial into the cell. In general, a composition includes a fluid. Forexample, the fluid contains a solution or a suspension, for example ofthe material, the cells, and other components.

In an embodiment of the method, the cell is a prokaryotic cell.Alternatively, the cell is a eukaryotic cell.

In general, introducing genetic material results in localizing thegenetic material to the nucleus of the cell. Alternatively, introducingthe genetic material results in localizing the genetic material into thecytoplasmic or non-nuclear parts of the cell, for example, into theendoplasmic reticulum, Golgi apparatus, mitochrondrion, and/or lysosomesor other membrane bound compartments in the cytoplasm. In a relatedembodiment, the method after or during localizing further involvesvisualizing the genetic material in the nucleus or in the cytoplasm ofthe cell using a detectable marker. For example, the detectable markeris an agent that is at least one of fluorescent, colorimetric,enzymatic, radioactive, and the like.

An embodiment of the method includes after passaging the mixture throughthe apparatus, dispensing the mixture into the receptacle.Alternatively, the method includes after passaging the mixture throughthe apparatus, and dispensing the mixture into a plurality ofreceptacles. For examples, passaging further involves dispensing themixture of the cell and the material at least once into the samereceptacle, i.e., the receptacle that originally housed the cells.Alternatively, the method involves passaging the mixture through theapparatus, and dispensing the mixture into each of a first receptacleand a second receptacle. An embodiment of the method involving a largenumber of receptacles is envisioned as automatically controlled, forexample, by robotics.

An embodiment of the method includes passaging the cell and the materialin the apparatus at least once for a period of time selected from atleast one of about: 0.1 minutes, 0.3 minutes, 0.5 minutes, 0.75 minutes,1 minute, 1.5 minutes, 2.0 minutes, 5.0 minutes, 7.0 minutes, 10.0minutes, 15.0 minutes, 20.0 minutes, and 30 minutes.

In various embodiments, the cell is a member of a population in aplurality of cells. In a related embodiment, the viability of the cellsis not substantially reduced, i.e., the efficiency of plating of cellsof the population remains substantially the same in comparison tocontrol cells not passaged, or control cells passaged absent material.

An embodiment of passaging includes dispensing the mixture of cell andthe material at least once into the receptacle, i.e., removing cellsfrom the receptacle into the apparatus and distributing cells into thereceptacle at least one once.

An embodiment of the receptacle is a centrifuge tube. An embodiment ofthe method includes passing or passaging the mixture of cells andgenetic material through a constriction in a continuous pipe having anentry point and an exit point. Such a system is used herein, forexample, in a flow-based, high throughput transfection system.

In various embodiments of the method, the fluid includes a Ca⁺²concentration that is less than about 200 nM. For example, thecomposition includes a Ca⁺² concentration that is less than about 150nM, less than about 100 nM, or less than about 50 nM.

An embodiment of the fluid includes a Mg⁺² concentration that is atleast about 1.5 mm. For example, the fluid, includes a Mg⁺²concentration of at least about 3 mM, at least about 10 mm, at leastabout 20 mm, or at least about 50 mm.

In related embodiments, the method further includes, after passaging,centrifuging the mixture to obtain a cell pellet and supernatant. Forexample, the method further includes removing the supernatant, addingcell culture medium to the receptacle, and re-suspending the cell pelletin the medium. In a related embodiment, the method further includesculturing the cells.

In related embodiments of the method, the cells are living postmitoticcells. For example, the postmitotic cells are neurons or sperm cells.For example, the neurons are ciliary ganglion neurons or dorsal rootganglion neurons.

Alternatively, the cells are living premitotic cells. For example, thepremitotic cells are at least one cell type selected from epithelialcells, hematopoietic cells, liver cells, and spleen cells.

In an embodiment of the method, the cells are physiologically inactive,such as inhibited by a chemical inhibitor, UV-inactivated, enucleated,anucleate, or heat-killed.

The material in some embodiments is a genetic material such as DNA orRNA. In a related embodiment, the DNA is cDNA. Alternatively, RNA is atleast one selected from mRNA, tRNA, rRNA, siRNA, RNAi, miRNA, and dsRNA.In an embodiment of the method, the fluid includes at least onetransfection agent. In related embodiments, the at least onetransfection agent is selected from: a nanoparticle, a liposome, a viralvector, a bacteriophage, and a detergent. For example, the transfectionagent is Lipolectamine.

An embodiment of the invention provides a kit for introducing a materialinto a nucleus of a living cell, the kit including the apparatus and/ortip assembly of any of the embodiments herein. In related embodiments,the kit further includes at least one of a receptacle, instructions foruse, and a transfection agent. For example, the transfection agent isselected from: a nanoparticle, a liposome, a viral vector, abacteriophage, and a detergent. For example, the transfection agent isLipofectamine.

An embodiment of the invention provides a method for introducing amaterial into at least one cell in a tissue or a monolayer of cells inculture, the method including: inserting the apparatus of any of theembodiments described herein into a receptacle, such that the receptaclecontains a composition including an effective amount of the material;and, contacting the at least one cell in the tissue or the monolayer inculture, such that contacting includes ejecting the composition underpressure onto the cell or cells, such that the material is introducedinto the cell in the tissue or the cells of the monolayer. In relatedembodiments the material is a genetic material, a protein, or a drug.

In general, ejecting the composition under pressure includes generatinga pressure wave having a particular frequency. The phrase “mechanicalwaves” refers to waves which propagate through a material medium (solid,liquid, or gas) at a wave speed that depends on elastic and inertialproperties of that medium. Wave motions of mechanical waves includelongitudinal waves and transverse waves. In general in the methodsherein, the pressure wave is a longitudinal wave, and the particledisplacement is parallel to the direction of wave propagation.

An embodiment of the method further includes, following contacting,observing the material entering the at least one cell without disruptingcell membranes or tissue. For example, observing includes analyzing cellmembranes using a microscope. In an embodiment of the method, the cellis a prokaryotic cell. Alternatively, the cell is a eukaryotic cell.

In general, ejecting the cells from the apparatus results in localizingthe material, e.g., the genetic material, to the nucleus of the cell.Alternatively, ejecting results in localizing the material tocytoplasmic parts of the cell for example, endoplasmic reticulum, Golgiapparatus, mitochrondrion, and lysosome.

An embodiment of the method includes visualizing the material with adetectable marker. In related embodiments, the detectable marker is anagent that is at least one of fluorescent, colorimetric, enzymatic, orradioactive. For example, the material if DNA encodes a fluorescentprotein, or the material if a protein includes a fluorescent tag.

In general, the cell is a plurality of cells. In an embodiment of themethod, the cell is a living cell within a population of living cells,and the viability of the cells is not substantially reduced.

In general, the genetic material is DNA or RNA. For example, the DNA iscDNA. For example, the RNA is at least one class of RNA selected fromthe group consisting of mRNA, tRNA, rRNA, siRNA, RNAi, miRNA, and dsRNA.The material in other embodiments is a protein or a polypeptide, such asinsulin, EGF, EPO, or IGF II.

In general, the tissue is a mammalian tissue. For example, the mammaliantissue is a human tissue, for example, skin, kidney, pancreas, liver,lung, heart, brain, spinal cord, bone marrow, and eye. An embodiment ofthe monolayer includes stem cells, for example, at least one stem cellselected from hematopoietic, hemangioblast, mesenchymal, hepatocyte,pancreatic, pulmonary, neural, fetal, and embryonic. The tissue may alsobe derived from other vertebrate species including mouse, rat, pig,goat, horse, cow, monkey, fish and bird. Alternatively the method mayalso be used with living cells from invertebrate species includingroundworm, molluscs, insects, echinoderms, and the like, or with livingcells from plant cells or from yeast.

The invention having now been fully described, it is further illustratedby the following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications cited throughout this application, are herebyincorporated herein by reference in their entireties.

EXAMPLES Example 1: Deficiencies of Prior Art Techniques

Methods to load antibodies into neuron cells (trituration) have beenattempted to reduce protein expression (Beerman et al., (1994) MethodsCell Biol 44: 715-732; Buchstaller et al., (2000) Microsc Res Tech48(2): 97-106; Diefenbach et al., (2002) J Cell Biol 158(7); 1207-1217;Diefenbach et al., In: Cells J E, Carter N, Simons K, Small J V, HunterT, Shotton D, editors. (2002) Cell Biology-A Laboratory Handbook. 3rdEdition (4 volumes). As antibody molecules are significantly smaller inmolecular mass and overall length than are sequences of plasmid DNA, andas a DNA molecule would have to pass through not only the plasmamembrane but also the inner nuclear membrane, these prior efforts havedemonstrated the limitations of getting such larger macromolecules suchas plasmid DNA into cells or into the nuclei of cells.

Trituration requires substantially higher velocities and changes inpressure for the significantly larger plasmid DNA to penetrate one ortwo membrane systems of the cell. Further, such higher velocities resultin loss of membrane integrity, and indeed trituration loading at highervelocities causes substantial cell death, the creation of cellulardebris from ruptured cells, introduction of air bubbles, anddifficulties in suitable design and method.

Examples 2: Design of an Apparatus to Introduce Genetic Material intoLiving Cells

To produce a device to introduce a material into living cells,borosilicate glass tubes were precision flame-polished at one end (tip)to reduce the size of the opening to less than or about 0.2 to about 0.5mm in diameter. Using this device, the cells were passed through asignificantly smaller diameter passageway than has previously been usedfor cell passaging, with a result that the cell velocity significantlyincreased as the diameter of the passageway narrowed. The narrowing ofthe exit tip assembly of the glass tube generated the greater fluidvelocities for introduction of the genetic material into the living,cells. Most important, the cells necessarily had undergone a rapidchange in fluid pressure as a result of the change in fluid velocity.The curvature of the inner surface of the narrowed portion of the fluidpath contributed to the cell pore formation and cellular membraneopenings. Further, the highly polished end was produced to prevent shearforces from forming as the cells encountered edges along the fluid path,therefore the polished end was observed to have enhanced cell viability.

Examples herein describe tip assemblies and methods of use forintroducing material into a cell, FIG. 1 panel A and FIG. 2 panel B arethree-dimensional representations of exemplary shapes of inner surfacesof tips having a top opening proximal to an apparatus that applies apressure such as a pump which impels a fluid, a channel portion leadingto a constriction portion, and a distal opening for releasing the fluid.The channel portion has a greater inner diameter and cross-section areathan the constriction portion.

FIG. 1 panel B and. FIG. 2 panel B are graphs of the eighth degreepolynomial function used to describe a plot of the tip assemblies ofFIG. 1 panel A and FIG. 2 panel B respectively. The graphs describe aradial distance of the tip from a center axis of the fluid path on theordinate, as a function of the length of the tip on the abscissa. Theplot shows the fluid path along a length that includes: the innersurface of the tip from the proximal opening to the channel portion andconstriction portion and to the distal opening.

Data in examples herein were obtained with neurons and non-neuronalcells using magnesium/calcium solutions generally having 1.5 mM Mg⁺²/200nM Ca⁺². A low Ca⁺² concentration in the solution was used to protectthe cells from calcium-induced cell death, and prolong the open state ofmembrane holes or pores. Neurons are known to lose viability followingprolonged Ca⁺² influx associated with activation of calcium-activatedproteases. Minimal signs of cell death were observed in the neuronalcultures monitored for a period of 48 hours following use of the methodsand these solutions.

Example 3: Introducing Genetic Material into Living Cells

Ciliary ganglia from chick embryos used in examples herein weredissociated from each other using trituration.

Cells were placed in a small volume (50 μl to 100 μl), and a solutionhaving a high concentration of plasmid cDNA (70 μg/ml) containing a genethat encodes green fluorescent protein (GEP) fusion proteins of interestwas added to the cells.

A very fine bore polished glass tube was then used to pass the cellsthrough the tip at high velocity. The fluid was passaged sequentiallyback and forth through the small bore tip about five to about ten times,into a vessel or receptacle, in this example a 1500 μl Eppendorfmicrocentrifuge tube. The small volume of cells and plasmid cDNA wasalmost entirely passaged, which maximized exposing the cells to the DNAduring the high velocity transitions. The passaging process wasperformed while monitoring volumes and minimizing production of bubbles,as extracellular oxygen is toxic to cells. As the cells were passedthrough the tip of the glass tube, pressure was gradually increased,which prevented bubble formation and improved cell survival among cellsin the population treated by the technique. A constant fluid pressurewas maintained during fluid passaging, to avoid sudden, instantaneouschanges in fluid pressure that damage cells by rupturing the cellularmembrane. The methods herein gradually increased or ramped up the fluidflow to avoid damaging the cells. The dampening effect of fluid responseto pressure changes also contributed to the ramping of increase in fluidflow. Sudden changes in fluid pressure to initiate or alter fluid flow,were carefully analyzed to determine effect of and thereby reduce damageto the cells for example to avoid rupturing of cell membranes.

The method for initial dissociation and high velocity passaging of thecells was performed in a solution that was substantially free of or hada low concentration of calcium, and that included a high concentrationof magnesium (from 1.5 mM to 5.0 mM). This solution was designed toprotect the cells, for example neurons, from calcium influx, calciumchallenges, and resultant calcium-mediated cell death through activationof calcium-dependent proteases. Such activation of calcium-dependentproteases is specific to the type of cell treated, and therefore higheror lower concentrations of calcium or magnesium were used in Examplesherein according to the type of cell.

Following passaging with nucleic acid, cells were collected bycentrifugation in a microcentrifuge at 3000 rpm, or any other rotationalrate sufficient to forming a pellet, and the supernatant was removedfrom the cell pellet, and was replaced with fresh culture medium. Thecell pellet was gently re-suspended in medium, and the cell suspensionwas cultured on plates. Expression of GFP gene and GFP fusion proteinwas monitored and these proteins were observed to have been expressed asfluorescence was observed within three to six hours in the cell bodiesof each of the neuronal and the nonneuronal cells found in the gangliapopulation. See FIG. 3 and FIG. 4.

A control vector constructed from Sindbis virus was used to tranfectcells, and recipient cells were observed to not express encoded proteinuntil at least about 12 hours after transfection of the cells. Themethods and devices herein were thus observed to have achieved visibleGFP expression, within a fraction of the lag time of the method usingSindbis virus, and lag in expression time was reduced by about 50% toabout 75%.

Example 4: Expression of Myosin Va

A gene encoding a GFP-myosin V fusion protein was introduced into chickciliary ganglion neurons. Movement of GFP-myosin Va-positive membranecompartments within axons and cell bodies was observed and thelocalization of GFP-myosin Va was consistent with cellular localizationof myosin V in control untreated cells in vivo. See FIG. 6 and Table 1below.

Myosin Va is a large multi-domain protein (190 kD), therefore observingrapid expression from a relatively long plasmid required of the genesencoding such a large protein, was surprising. Expression of GFP-myosinVa was initially observed using the methods and devices herein withinthree hours, and by six hours punctate or localized compartments offluorescence were observed.

These data show that expression of the myosin was associated withdirected motion that is typical of Membrane compartments, the motionconveyed by the motor activity of myosin Va or associated motor proteinssuch as kinesin, which bring myosin Va-linked membrane compartments tothe periphery along microtubules. Distribution and amount of expressionof the GFP-myosin Va construct and the GFP construct each increased inabundance slowly during 24 hours following introduction of theconstruct.

Example 5: Kinetics and Localization of Expression

Expression of GFP was observed to increase slowly for 24 hours aftertransfection of the cells with constructs using the methods and devicesherein. At 24 hours, GPF protein was observed to have been expresseddiffusely throughout the cell, and was also observed inintensely-fluorescent membrane compartments within the cytoplasm of thecell. See FIG. 3.

The frequency (number of cells randomly sampled, and percent) of GFPprotein control expression (GFP) or GFP-myosin Va construct (Myosin Va)expression in neuronal and nonneuronal cells, observed as a function oftime after transfection using the method herein, including plating thecells in culture, is shown in Table 1. It was observed that nonneuronalcells required a greater time period for appreciable expression, andthat neurons show consistently high frequencies of expression in eithera diffuse form early after culturing, or in the form of punctate orcompartmentalized staining later after culturing. Without being limitedby any particular theory or mechanism of action, it is envisioned thatthe observed staining is related to a relatively high metabolic activityof neurons growing in culture.

TABLE 1 Frequency in cells of gene expression observed as a function oftime Time (h) after GFP GFP Myosin Va Myosin Myosin culturing staintotal GFP % stain total Va % Neurons 3 5 5 100 17 17 100 4 18 18 100 1515 100 5 23 26 88.5 7 44 45 97.8 30 30 100 21 22 22 100 Nonneuronalcells 3 0 4 0 5 18 27.8 4 3 6 50 0 2 0 5 11 20 55 7 26 32 81.2 21 3756.8 21 33 35 94.3

Expression of GFP protein in nonneuronal and neuronal cells wasdetermined to achieve a maximum at 48 hours after the time ofloading/introduction (See FIG. 4). GFP protein expression was observedto be abundant in compartments in the cytoplasm, and was mostly absentfrom the nucleus. FIG. 4.

Strong evidence of localization of myosin Va was observed both in thecell body and the neuronal processes. Localization was observedparticularly in branch points and tips of growing axonal processes. FIG.5 Panel A and Panel B. Data in Examples herein demonstrate successfultransfection of neuronal cells with genes encoding different myosin Vaconstructs, with substantial expression observed in the cells.

Transfection was observed to result in specific changes in nerve cellprojections at the growing tips. Changes were observed in neuronalfilopodia, the finger-like projections of growing tips (FIG. 6).Transfection of enhanced GFP-Va tail was observed to result in a slightbut significant reduction in filopodial length (p<0.01, Student'st-test) at 15 hour compared to eight hours. Number of filopodia/growthcones for each construct group include: control GFP (538/59), enhancedGFP-myosin-Va (630/69), and enhanced GFP-Va tail (653/76). The data showthat overexpression of myosin-Va resulted in increased filopodiallengths, and expression of a truncated form of myosin-Va, which does notbind actin filaments, resulted in reduced filopodial lengths. These datashow successful transfection and function of transfected myosin-Vaconstructs in primary neurons using the methods and devices herein, andexpression of differential phenotypes related to functions of theencoded genes.

Example 6: Expression of Genetic Material in Mouse Sperm Cells

The methods and devices herein were applied to a cell system, the maturesperm cell, in which introduction and expression of genetic material hasnot heretofore been observed. Sperm cells are notoriously difficult topermeate, and have been characterized as having no nuclear translationand very little RNA. Protein synthesis in sperm cells is confined tomitochondria within the small amount of nature sperm cytoplasm. Thus,sperm cells have largely been considered inaccessible to methods ofrecombinant DNA technology.

The methods and devices in Examples herein were used to introduce aplasmid encoding pCherry, a variant of GFP protein, that fluoresces inthe red part of the visible spectrum (Shaner et al., Nat Biotechnol22(12); 1567-1572, 2004). The pCherry gene is operatively linked to andcontrolled by a CMV promoter. Following transfection the living mousesperm cells were incubated overnight at 37° C. pCherry fluorescence wasobserved within 24 hours in a subset of treated sperm cells (FIG. 7).These data show for the first time successful expression of arecombinant gene, encoding pCherry protein, in the sperm cellstransfected by the methods herein.

Localization of fluorescence of GFP-positive sperm cells was comparedwith that of a set of control sperm cells treated with a membranepermeable fluorescent dye, Lavacell, which permeates cells and stainsinternal membranes. Fluorescence of Lavacell stained sperm cells wasobserved throughout the cell including the cell head, midpiece and tail.See FIG. 7.

In contrast, transfected pCherry expressing sperm cells (not treatedwith Lavacell) showed essentially no fluorescence in the head portion ofthe cells. These cells were observed to have intense fluorescence in themidpiece where mitochondria are abundant, with minimal fluorescence inthe flagellum or tail segment (FIG. 7). pCherry expression was observedin 75% of the sperm cells at sites of localization, consistent withlocalization of RNA in sperm cells.

Example 7: Introducing Genetic Material into Cells using PressurizedSolutions

Genetic material is introduced into a cell in a tissue, or in amonolayer in culture, by ejecting a composition including a geneticmaterial onto the cells in the tissue or monolayer. The genetic materialis introduced into the cell/monolayer of cells using fluid pulsed athigh velocity which creates conditions similar to that of the methodsdescribed herein, such that the increase in fluid speed corresponds to asimultaneous decrease in pressure at the cell membrane. The pulsed fluidmomentarily stretches the cell membrane (Bernoulli's principle) and apore or a plurality of pores is formed that acts as a point of entry forthe genetic material.

The fluid containing the genetic material is pulsed at a specificpressure, frequency, or force to form a pressure wave. Alternatively,the fluid is pulsed across the surface of, or directly at the face of,the tissue or monolayer.

It is envisioned that genetic engineering in situ of tissues needing atherapeutic gene is achieved by methods herein, for example, a geneencoding a normal allele of a defective inherited gene, for example,into a hematopoietic tissue such as bone marrow or liver, is deliveredby this method.

Example 8: Expression of Genetic Material in Human Endothelial Cells

Human umbilical vein endothelial (HUVEC) cells similar to most primarycells, are characterized by poor transfection rates using previouslyknown transfection methods such as nucleofection, electroporation, andusing current transfection products such as lipofectamine The methodsand tip assemblies herein were used to transfect HUVEC cells with aplasmid encoding a fusion protein of enhanced green fluorescent protein(EGFP) and human porcine endogeneous retrovirus receptor (HuPAR2), aprotein that is specifically localized in perinuclear subcellularmembranous compartments.

Examples herein generated an enhanced GFP (EGFP)-tagged C-terminalHuPAR-2 fusion protein (HuPAR-2/EGFP). The HuPAR-2 open reading frame(ORF) was amplified from the Tope-pCRII clone by using the primers5′-ACGCGGTACCCAGGGGTCTACACAGTCCTTT-3′ (SEQ ID NO: 1) and5′-ACGCAGATCTAGCATCTTTGGACCTACCTAG-3′ (SEQ ID NO: 2), which contain KpnIand BglII restriction sites. The product was cloned into Topo-pCRII(Invitrogen Life Technologies) and excised using KpnI and BglII. Thisfragment was cloned upstream and in-frame of the EGFP ORF in the KpnIand BglII fragment of the EGFP fusion vector pEGFP-NI (BD BiosciencesCLONTECH; San Jose, Calif.).

HUVEC cells were grown in ATCC endothelial cell media and were contactedwith a vector (35 μg/ml; 7005 base pairs) encoding a fusion protein ofEGFP and HuPAR2 suspended in Hank's balanced salt solution (HBSS;Sigma-Aldrich, St. Louis, Mo.). The cells were plated on a glass bottom35 mM petri dish for 15 minutes to settle and to attach to the surfaceof the dish. Growth medium was added and the cells were incubated for 24hours at 37° C. and 5% CO₂.

The cells were visualized using a laser scanning confocal fluorescencemicroscope and contrast-enhanced version of brightfield microscopy,namely DIC (differential interference contrast optics), and fluorescencewas analyzed in ten randomly chosen microscope fields 24 hours aftertransfection. Total number of HUVEC cells and number of cells showingGFP-HuPAR2 fluorescence were determined, and the percentage of HUVECcells with GFP-HuPAR2 fluorescence was calculated and compared (Table2). Table 3 shows a statistical analysis of the data.

Photomicrographs of the HUVEC cells were analyzed using fluorescencemicroscopy. It was observed that an average of greater than 70%transfection efficiency was achieved for the cultured HUVEC cells. SeeTable 2 and Table 3. Representative DIC photomicrograph data andfluorescence photomicrograph data for a single cell and for a pluralityof cells are shown in FIG. 8 panels A and B, and these data show thatthe cells were effectively transfected cells. Dark granules wereobserved by DIC in the membranous compartments surrounding the nucleusof a single HUVEC cell (FIG. 8 panel A left photomicrograph) and aplurality of HUVEC cells (FIG. 8 panel B). HUPAR2 protein localizesspecifically in a perinuclear subcellular membranous compartments, andcells observed herein showed significant GFP fluorescence staining inthe perinuclear subcellular compartments of the cells transfected withthe GFP-HuPAR2 plasmid using methods and systems herein. FIG. 8 panel Amiddle photomicrograph. An overlay of the DIC photomicrograph and thefluorescence microscope photomicrograph showed that the GFP-HuPAR2 waslocalized specifically in the perinuclear membranous compartments. Thesedata show HUVEC cells were successfully transfected using the EGFP-HuPAR2 construct and the methods and the tip assembly herein.

TABLE 2 HUVEC cells show GFP-HuPAR2 fluorescence in 10 randomly chosenmicroscope fields 24 hours after transfection HUVEC cell GFP-HuPAR2percent HUVEC cells number per field fluorescent cell number withfluorescence 10 7 70 16 12 75 10 8 80 18 17 94.4 16 8 50 13 7 53.8 20 1785 16 12 75 9 4 44.4 15 12 80

TABLE 3 Statistical analysis of data shown in Table 2 standard standardaverage deviation error (x) (sd) (se) total number of HUVEC cells 14.33.68 1.16 HUVEC cells showing GFP-HuPAR2 10.4 4.35 1.37 fluorescencepercentage of total number of HUVEC 70.8 16.26 5.14 cells that areHu-PAR2 fluorescent cells

As the cells in this Example were transfected with a previously frozenvector, which was a large plasmid (7005 base pairs), the methods and tipassemblies herein were surprisingly more effective in transfecting andtransducing cells with a genetic material and under circumstances thatby other methods would have proven to be much less efficient.

Example 9: Construction and Adaptations of Tip Assemblies

Tip assemblies suitable for use in methods herein include thoseconstructed of a variety of different types of materials and indifferent sizes and shapes. Fluids having cells and materials areintroduced into the tip assemblies, and the effectiveness of the tipassemblies to introduce material into cells is determined by one skillin the art of cell transfection.

Adaptation of design of the tip assemblies includes producing structureswith varying fluid path shapes, number of constriction portions, and byvarying concentration of materials in the composition including thepresence of organic and inorganic agents.

Tip assemblies can be used with various known types of reservoirs andflow devices. For example the reservoir is a centrifuge tube, a bin, abag, or a bottle, and the flow device is a hand-held 200 μL pipette orhand pump. Each of the tip assemblies is adapted by obtaining data(e.g., pressure differentials, cell membrane porosity and cellviability) for each of the multiple tip assemblies and each of thevarying concentrations of substances in the tip assemblies.

FIG. 9 is a drawing of a three-dimensional representation of anexemplary tip assembly having a body including an opening shown at thetop which is proximal, proximal opening 900, to the reservoir and thefluid flow device, an attachment portion 902, a tip assembly shoulder901 for ejecting the tip assembly when attached to a flow device, achannel portion 903, a constriction portion 904, and a distal opening905 shown at the bottom. The fluid path direction proceeds from theproximal opening 900 to the distal opening 905. The channel portion 903has a greater inner diameter and cross-section area than theconstriction portion 904. The tip assembly shoulder 901 extendslaterally from an outward surface of the attachment portion 902. A lowerejector section of an embodiment of the flow device removes the tipassembly from the flow device.

To operate, a flow device or fluid handling device in certainembodiments is attached to the tip assembly at the attachment portion.The fluid containing cells and material is drawn into the tip assemblyfrom the distal opening through the constriction portion to the channelportion of the tip assembly. The top meniscus of the fluid surface andthe end of the flow device are separated by a distance sufficient toavoid contact between the fluid with the flow device, so that differentmaterials can be used in each tip. The fluid device is used to impel thefluid through the channel portion to the constriction and the distalopening, and a reduced pressure in the constriction portion compared tothe channel portion is achieved, forming pores in cells containingwithin the fluid, and a material included in the fluid is introducedinto the cells. In certain embodiments, a fluid containing the cells isdrawn into a longer fluid path such that sufficient fluid velocities areattained when passing through the constriction portion of the tipassembly.

The tip assemblies are evaluated under conditions and by the methodsused in Examples above. The data obtained are used to determine cellviability and presence of material in the cell. Data show that the tipassemblies introduced material into cells by reducing the fluid pressureof the fluid and by increasing the membrane porosity of cells, in livingcells and non-dividing cells. These tip assemblies were shown byExamples herein to be more effective, more efficient and convenient forintroducing material into cells than previous methods and devices.

Example 10: Design of an Embodiment of the Transfection System

An embodiment of the transfection system described herein is shown inFIG. 10 panels A-D. The main components of the device are a tipassembly, a computer programmable syringe pump 101, 102 connected to thetip assembly by means of a USB converter 103 and an RS232 interface 104,and a power supply 110. As shown in FIG. 10 panel A the syringe pump101,102 is attached to the inner side of the metal lid 105 of a jump box106. The USB converter 103 and the RS232 interface 104 that connects thesyringe pump to an external computer are also attached to the inner sideof the metal lid 105 of the jump box 106. The hardware is not visibleduring use of the device in laboratory. An external computercommunicating through the RS232 interface 104 and the USE converter 103is used for controlling the syringe pump 101,102 using the data analysissoftware MATLAB (Mathworks, Natick, Mass.). Alternatively thetransfection device uses a simple graphical user interface software,LabWindows/CVI Run-Time Engine 8.5.1 (National Instruments, Austin,Tex.).

The position of the programmable syringe and the pump mechanism 101,102on the jump box 106 right is shown in FIG. 10B. The borosilicate glasssyringe 101 has a UHMWPE (ultra-high molecular weight polyethylene) seal107 for lubrication and solvent resistance. A TYGON® tube 109 extendsthrough the metal lid 105 of the jump box 106 and connects to theproximal end of a soda lime glass capillary tube (the tip) throughadditional tubing. The capillary tube is an exemplary tip (FIG. 1 panelA) having the dimensions: inner diameter 1.1-1.2 mm, wall thickness 0.2mm, length 75 mm.

The power supply 110 is located inside the jump box 106 to the lowerleft (FIG. 10 panel C). The power supply 110 is a single phase 24V, 2.5Apower supply (PHOENIX CONTACT GmbH & Co, KG, Step Power, BlombergGermany), standard for distributor boards and flat control panels. Thepower supply (110) has low standby losses and high efficiency.Alternatively, a power adaptor is FSP60-11 (FSP North America), having24V, 2.5A output. The power sources take 100-240V, 2.0A, 50-60 Hz input.

As shown in FIG. 10 panel D, the jump box 106 is located in a tissueculture flow hood for sterile conditions. During use the metal lid 105remains in the closed position and the outer lid in an open position111.

Example 11: Fluid Cycling Parameters during Transfection

This example illustrates the general flow parameters during fluidcycling through the tip of the tip assembly used in the methods andsystem or devices described herein.

A mixture containing cells and a composition to be introduced into thecells in a fluid is drawn into and forced out of the tip of thetransfection device herein iteratively, resulting in cycles of inflowsand outflows. An execution stroke followed by a return strokeconstitutes a cycle. The return stroke and the execution stroke operateaccording to the same parameters. The programmable syringe 101,102described in Example 10 produces the flow cycles. A schematic drawingillustrating movement of the syringe plunger 108 in an embodiment offluid cycling is shown in FIG. 11, with time represented on the abscissaand the position along the length of syringe at which the plunger islocated at that time during a cycle represented on the ordinate.

The following command sequence was used in this embodiment:/1L14v400V900c200A500M200gL20v100v1000c1000A400M100A0G8L14v400V900c200A0R

The plunger motions and associated parameters in this command sequenceproceeding from left to right are described herein. The plungerpositions in the command sequence correspond to plunger positionsindicated on the ordinate in FIG. 11 with the letter A added as aprefix. For example, plunger position 500 in FIG. 11 is position A500 inthe command sequence. FIG. 11 illustrates eight cycles which wereperformed as follows:

L14 is the initial velocity ramp (acceleration), v400 is the startingvelocity and V900 is the maximum velocity during this ramp. A500 is theposition the syringe plunger (108) moves to initially, loading the tip.A500 is called the load position. c200 is the cutoff velocity when theplunger reaches A500. M200 is a 200 milliseconds (ms) pause at the loadposition. The cycling begins after a 200 ms pause, indicated by g in thecommand sequence. Ramps of a cycle, excluding the initial velocity rampwhich has an acceleration L14, have a maximum acceleration L20, astarting velocity 100 (v100), a maximum velocity 1000 (v1000) and afinal or ending velocity 1000 (c1000). A downward movement to positionA400 reduces the fluid volume to a less than an initial fill volume,leaving a cycling volume in the tip. M100 indicates a 100 ms pause afterwhich there is a ramp from position A400 to A0. This represents the endof the first cycle and is indicated by G in the command sequence. Thenumber 8 indicates that this command sequence has 8 cycles. The firstcycle differs from subsequent cycles as it includes the loading step. Atthe end of the command sequence is a cycle with velocity ramp L14,starting velocity v400, maximum velocity V900, end position A0, andfinal velocity c200. The letter “R” at the end represents an executecode.

In the examples described herein similar cycles were used with cells andplasmid DNA or siRNA or miRNA.

Example: 12 Introduction of EGFP-CDC42 Plasmid into HUVEC Cells

The methods and system or device herein were tested to transfect aprimary cell which are more delicate and usually more vulnerable totransfection than transformed cells that have been propagated in thelaboratory for many years. Primary HUVECs (human umbilical veinendothelial cells) were chosen for this purpose.

HUVECs were transfected with a plasmid encoding an EGFP-CDC42 (enhancedgreen fluorescent protein-cell division control protein 42) fusionprotein. The plasmid concentration mixed with cells was 70 μg/ml. Cellswere allowed to become confluent by culturing them for two days prior totransfection, trypsinized to detach the cells from the cell culturesubstrate and suspended in fluid for transfection.

Transfection was performed using the parameters: fluid acceleration 6μl/s/s, flow rate 160 μl/s, cycling volume 50 μl, and two continuoussets of cycles each set having 25 inflows and outflows of fluid throughthe tip impelled by a 2.5 ml syringe. A volume of 63 μl contained500,000 cells and the plasmid.

Images of cells taken with a laser scanning fluorescence microscope 24hours after transfection showed punctate (or spotted) subcellularEGFP-CDC42 fluorescence. The fluorescence was cytoplasmic and in theperiphery of the cell, consistent with CDC42 localization, and wasexcluded from the nucleus (FIG. 12 panel A). Fluorescent cells in twentyrandomly selected 20× magnification microscope fields were sampled.Results showed that a pooled average of 88.2% (135/153) of the cellsdisplayed a CDC42 like EGFP fluorescence. Transfection using AMAXANucleofactor™ (Lonza Cologne GmbH, Cologne, Germany) electroporationunit was tested for comparison and either no transfection or lowertransfection efficiency (less than 30%) was observed.

Images of transfected HUVEC cells with EGFP-CDC42 plasmid using themethods and the transfection device herein include one of EGFPexpression in cells that had divided before imaging (FIG. 12 panel B).Consistent with the expression pattern of a protein with a role in celldivision EGFP fluorescence was localized to a region between thedaughter cells.

EGFP fluorescence of HUVEC cells transfected with. EGFP-CDC42 usingmethods and the transfection device described herein was observed atnormal PMT sensitivity and compared with that observed at higher PMTsensitivity (FIG. 12 panel C and D). At normal PMT sensitivity most ofthe fluorescence was observed to be localized to large granules (FIG. 12panel C). At higher PMT sensitivity EGFP-CDC42 expression was detectedin a subset of cells within numerous subcellular granules which weresmaller in size (FIG. 12 panel D). Scanning at a higher PMT sensitivitywas observed to offset potential autofluorescence observed in the largegranules at normal PMT sensitivity. The fluorescence in the smallergranules was tested by photobleaching using high-sensitivity laser lightin scanning mode and was observed to be specific to the expression ofEGFP-CDC42 fusion protein.

The example above demonstrates that the methods and devices describedherein not only led to efficient transfer of the plasmid into a primarycell, the plasmid was transcribed into mRNA, the mRNA translated intoprotein and the protein was expressed with correct localization, therebyleading to a successful transfection.

Example 13: Introduction of 7 kb EGFP-Actin Plasmid into HUVEC Cells

Success in transfection can also be tested using measurement of mRNAcopy number following transfection. Measurement of mRNA copy number isuseful in situations such as when the fluorescence signal is fainteither due to the peculiar nature of the fluorescent protein beingexpressed or when there is a high autofluorescence background.

Primary HUVEC cells from P5 mouse were transfected with a 7 kb plasmidencoding EGFP-actin fusion protein using methods and device describedherein to observe expression of EGFP-actin in actin filament componentof the cell cytoskeleton. 500,000 cells were suspended in 100 μl ofphosphate buffered saline in a 1500 μl centrifuge tube to which theEGFP-actin expression plasmid at a final concentration 70 μg/ml wasadded and mixed with the cells.

Parameters used for transfection were: fluid acceleration, 6 μl/s/s,flow rate, 160 μl/s, cycling volume 60 μl, volume of syringe used, 2.5ml, and sequential passage cycles consisting of three consecutive setsof 25 cycles of fluid motion through the tip. Cells were grown inculture for 48 hours and mRNA production resulting from the transfectionwas assessed using a method that determines the exact copy number ofmRNA per cell. Shih et al. 2005, Exp Mol Pathol 79:14.

The results are shown in FIG. 13 panels A, B and C. Cells transfectedwith the transfected device herein are labeled as TC in FIG. 13 C. As acontrol cells were also subjected to the transfection protocol in theabsence of the plasmid. These cells were also examined for EGFP-actinmRNA copy number (labeled CT in FIG. 13 panel C). Cells were alsotransfected using the AMAXA Nucleofector™ electroporation unit and arelabeled as AMAXA in FIG. 13 panel C. Cells transfected using the AMAXANucleofector™ electroporator resulted in the generation of an average of1152.75 copies of mRNA per cell against a background of only 0.08average copies of mRNA per cells for the control cells. In contrast,cells transfected with the transfection device resulted in an average of3652.66 copies, of EGFP-actin mRNA per cell (FIG. 13 panel C). Inanother experiment performed under similar conditions, comparableaverage mRNA copy numbers were observed (1152.75 copies of mRNA per cellfor transfection with AMAXA Nucleofector™ electroporator versus 3853.95copies of mRNA per cell for transfection with the transfection devicedescribed herein). These results show that transfection using thetransfection device described herein resulted in dramatically increasedcopy numbers of EGFP-actin mRNA per cell and the increase wasreproducible.

Thus mRNA copy number measurements also confirmed that methods anddevices described herein are useful to achieve efficient transfectioneven in a primary cell.

Example 14: Introduction of Genetic Material into Jurkat Cells

Jurkat cells, a cell type known to be refractory as transfectionrecipients were tested for transfection efficiency with EGFP (Addgene,Cambridge, Mass.) using the methods and the apparatus herein.Approximately 250,000 cells were suspended in 50 μl of transfectionmedium, HBSS (Hanks Balanced Salt Solution, Invitrogen Carlsbad, Calif.)absent calcium or magnesium and contained 0.2% w/v EDTA (ethylenediaminetetraacetic acid). As control a mock transfected sample was transfectedabsent plasmid. Other transfections contained 2.0 μg (55 μl total volumewith cells, 36 μg/ml), 5.0 μg (62.5 μl total volume with cells, 80μg/ml) or 10.0 μg (75 μl total volume of cells, 130 μg/ml) of plasmid.Transfection parameters were: fluid acceleration 6 μl/s/s, flow rate 160μl/s, cycling volume 60 μl using the 2.5 ml syringe and continuouscycling consisting of 25 inflows and outflows through the tip. Cellswere grown in culture for 48 hours and EGFP fluorescence (in FL1-Hchannel) was quantified using flow cytometry (FIG. 14).

It was observed that 10% of the cells showed 50 to 90% of maximumintensity of EGFP fluorescence. Data obtained from cells treated with5.0 μg of plasmid showed EGFP fluorescence higher than the backgroundlevel observed in the control transfection absent plasmid. Fluorescenceintensities observed using either 2.0 or 10.0 μg of plasmid were at thebackground level.

Example 15: Transfection of Primary Mouse Brain Astrocytes using theTransfection System herein

The methods and transfection system or device described herein, withsome modifications were used to test transfection of another type ofprimary cells.

Primary astrocytes (1 to 2 million cells) were isolated from mouse brainand suspended in DMEM culture media (Dulbecco's modified Eagle medium,Invitrogen, Carlsbad, Calif.) and kept in a cell culture incubator(humidified, 37° C. and 5% CO₂) until transfection. For transfectioncells were centrifuged and resuspended in 100 μl of HBSS (Ca²⁺/Mg²⁺free) containing 20 μg/ml of EGFP control plasmid (Invitrogen, Carlsbad,Calif.) or 20 μg/ml of Cy3-labeled miRNA (AM17011, 25 base pair designedRNA oligonucleotide, Ambion, Austin, Tex.)).

The cells were transfected using a tip prepared as follows. A customfabricated flint glass tube having an ID of 0.88 mm, an OD of 1.23 mmand with wall thickness of 0.14 mm was heated to obtain the curvature asdescribed herein. A further additional microchannel, 250 μm long with anarrowing almost cylindrical portion nearest to the opening of the tipwas added to the configuration of the tip. The tip culminated in a smalltip opening, diameter 175-200 μm. (FIG. 1 panels C and D).

Transfection with EGFP plasmid was performed using a tip, 200 μmdiameter, and transfection with Cy3-labeled miRNA was performed using atip, 175 μm diameter.

The mixture of cells and the EGFP plasmid or the miRNA were passagedthrough the tip iteratively for 60-100 times using the methods andtransfection device described herein. The mixture was drawn into the tipin a cycling volume of 60-80 μl. In view of the smaller diameter of thetip opening used in this transfection, cycling parameters were modifiedfrom those used in Examples 12-14, described generally in Example 11.Only the inflow parameters were changed. Inflow velocity was reduced to50-80 μl/s and acceleration was reduced to 1-4 μl/s in order to permitunimpeded entry of cell suspension into tip without clogging. Thusassymetric cycles of slower inflow and maximum outflow velocity (240μl/s) were utilized. After transfection cells were cultured andincubated in a cell culture incubator for three days, then fixed andmounted on glass slides for imaging using a confocal microscope.

Transfection of the primary astrocytes using a tip assembly withnarrower tip opening and containing an additional microchannel at theproximal end (FIGS. 1C and D) led to successful transfer of EGFP plasmidor the Cy3-labeled miRNA into the cells. Representative images observed3 days after transfection are shown in FIG. 15 panels A-F. Panels A andB show cells with varying levels of EGFP fluorescence intensities. PanelC shows a single bright fluorescent cell among a cluster of cells withfluorescence intensities equal to or only slightly higher thanbackground fluorescence. Fig. D is an image showing an overlay of afluorescence image of a transfected primary astrocyte over a brightfieldimage of the same cell. The image shows a highly fluorescent transfectedcell spread out on a culture substrate having a morphologycharacteristic of primary mouse brain astrocytes.

Transfection efficiency measured one day after transfection by observingcells having fluorescence above background was determined to be 13.8%(237 cells from 5 different microscope fields). The efficiency wasobserved to increase to 29.8% three days after transfection (188 cellsfrom 13 different microscope fields).

Panels E and F show transfected primary mouse brain astrocytes that haveeither a moderate level (panel E) or a high or background level (panelF) of internalized Cy3-labeled miRNA in the cytoplasm of the cells. ThemiRNA is excluded from the nucleus. The labeled miRNA appear as tinyround fluorescent spots. Transfection efficiency measured 3 days aftertransfection by observing cells that had fluorescence above backgroundwas determined to be 40.9% (41 cells from three microscope fields).

This example further demonstrates the applicability of the methods andtransfection device described herein for successful transfection ofprimary cells, thereby extending the utility of the methods and devicesherein.

What is claimed is:
 1. A method of introducing a composition in a fluidinto cells, the method comprising: a) passaging a suspension of cells ina fluid with the composition through a decrease in pressure, wherein thedecrease in pressure occurs in a device comprising a channel with atleast one constriction; b) repeating step a) one or more times, whereindecreases in pressure enhances temporary permeability in the cells,thereby introducing the composition into the cells.
 2. The method ofclaim 1, wherein the passaging of the suspension of cells in the fluidis performed continuously.
 3. The method of claim 1, wherein thepassaging of the suspension of cells in the fluid is performed with aplurality of pauses.
 4. The method of claim 3, wherein each of theplurality of pauses are from about 1 ms to about 100 s in length.
 5. Themethod of claim 1, wherein the decrease in pressure is from about 0.1%to about 95% of the pressure of the suspension of cells in a fluid withthe composition prior to passaging.
 6. The method of claim 1, whereinthe constriction has a cross-sectional area bounded by a shape selectedfrom the group consisting of circular, ellipsoidal, rectangular, orsquare.
 7. The method of claim 1, wherein the passaging is for a periodof time of about 1 second to about 30 minutes.
 8. The method of claim 1,further comprising observing localization of the composition to at leastone subcellular compartment selected from the group consisting of anucleus, a mitochondrion, a Golgi body, a chloroplast, a chromoplast, anendosome, a vesicle, a lysosome, an axon, a cytoplasmic membrane, anuclear membrane, and a cytoplasm.
 9. The method of claim 8, whereinobserving the localization of the composition comprises visualizing thecomposition with a detectable marker selected from the group consistingof a fluorescent marker, a chemiluminescent marker, a colorimetricmarker, an enzymatic marker, and a radioactive marker.
 10. The method ofclaim 8, wherein observing the localization of the composition furthercomprises quantifying directly amount of the composition that enteredthe cell.
 11. The method of claim 1, further comprising dispensing thesuspension of cells in the fluid and the composition into a receptacle.12. The method of claim 1, wherein the cells comprise a population of aplurality of living cells.
 13. The method of claim 12, wherein cellviability of the population of a plurality of living cells is from about1% to about 95% of control cells not passaged through the channel withat least one constriction.
 14. The method of claim 1, wherein the fluidcomprises a Ca²⁺ concentration from about 50 nM to about 500 nM.
 15. Themethod of claim 1, wherein the fluid comprises a Mg²⁺ concentration fromabout 100 nM to about 10 mM.
 16. The method of claim 1, furthercomprising the steps of: a) centrifuging the suspension of cells in afluid with the composition to obtain a cell pellet and a supernatant; b)removing the supernatant; c) adding a culture medium; d) re-suspendingthe cell pellet in the culture medium; and e) culturing the cells. 17.The method of claim 1, wherein the cells comprise a cell type selectedfrom the group consisting of epithelial cells, hematopoietic cells, stemcells, spleen cells, kidney cells, pancreas cells, liver cells, neuroncells, glial cells, smooth or striated muscle cells, sperm cells, heartcells, lung cells, ocular cells, bone marrow cells, fetal cord bloodcells, progenitor cells, peripheral blood mononuclear cells, leukocytecells, lymphocyte cells, living postmitotic cells, physiologicallyinactive cells, inhibited cells, UV-inactivated cells, enucleated cells,anucleate cells, heat-killed cells, non-reproducing cells, and syntheticcells having an artificial membrane.
 18. The method of claim 1, whereinthe composition comprises an inorganic compound, a drug, a geneticmaterial, a protein, a carbohydrate, a synthetic polymer, or apharmaceutical composition.
 19. The method of claim 18, wherein thecomposition comprises a genetic material.
 20. The method of claim 19,wherein the genetic material comprises a DNA or an RNA.
 21. The methodof claim 18, wherein the composition comprises a protein.
 22. The methodof claim 1, further comprising assaying transfection of the cells. 23.The method of claim 1, further comprising applying to the suspension ofcells in the fluid and the composition at least one treatment selectedfrom the group consisting of an electric field, light comprising atleast one wavelength, and a sound pulse.
 24. The method of claim 1,wherein the fluid further comprises at least one transfection agentselected from the group consisting of a nanoparticle, a liposome, aviral vector, a bacteriophage, and a detergent.
 25. The method of claim20, wherein the RNA is selected from the group consisting of mRNA, tRNA,rRNA, siRNA, RNAi, miRNA, and dsRNA, or a portion thereof.
 26. Themethod of claim 1, wherein the step b) comprises up to 100 passagesthrough a channel comprising at least one constriction.
 27. A method ofintroducing a composition in a fluid into cells, the method comprising:passaging a suspension of cells in a fluid with the composition througha decrease in pressure, wherein a diameter of the constriction isgreater than a diameter of the cells in the fluid, wherein the decreasein pressure enhances temporary permeability in the cells, therebyintroducing the composition into the cells, wherein the change inpressure is created by a device comprising of a channel with at leastone constriction.
 28. A method of introducing a composition in a fluidinto cells, the method comprising: passaging a suspension of cells in afluid with the composition through a decrease in pressure, wherein thedecrease in pressure is created by a device comprising of a channel withat least one constriction, wherein the geometry of the channelcomprising at least one constriction is defined in two dimensions by theformula:f(x)=p1x ⁷ +p2x ⁶ +p3x ⁵ +p4x ⁴ +p5x ³ +p6x ² +p7x+p8 wherein x is aradial distance from a center axis of the channel to an inner surface ofthe at least one constriction and coefficients with 95% confidencebounds in parentheses comprise: p1 is −2.611e⁻¹⁶ (−6.043e⁻¹⁶,8.206e⁻¹⁷), p2 is 3.954^(e−13)(−3.195^(e−13), 1.11^(e−12)), p3 is−1.845^(e−10)(−7.821^(e−10), 4.131^(e−10)), p4 is1.662^(e−08)(−2.394^(e−07), 2.726^(e−07)), p5 is7.537^(e−06)(−5.186^(e−05), 6.694^(e−05)), p6 is −0.002137 (−0.009375,0.005101), p7 is −0.003185 (−0.4114, 0.4051), and p8=268.6 (261, 276.3),wherein the decrease in pressure enhances the uptake of the compositioninto the cells by increasing temporary permeability of the cells,thereby introducing the composition into the cells, and wherein thepassaging of the suspension of cells is substantially free of shearforces.
 29. A method of introducing a composition in a fluid into cells,the method comprising: passaging a suspension of cells in a fluid withthe composition through a decrease in pressure, wherein the decrease inpressure enhances the uptake of the composition into the cells byincreasing temporary permeability of the cells, thereby introducing thecomposition into the cells, wherein the change in pressure is created bya device comprising a channel with at least one microchannelconstriction, wherein the ratio of a length of the at least oneconstriction to a width of the at least one constriction is from 0.5:1to 2.5:1, and wherein the cells in the suspension are viable afterintroduction of the composition.